INTRODUCTION - 筑波大学e-archive.criced.tsukuba.ac.jp/data/doc/pdf/2008/12/Protocol text.pdf ·...

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INTRODUCTION

The word of “Bioprospection” can be defined as activities to explore useful

materials from bioresource, one of the most prospective renewable resources in the

world. Throughout the history, human beings have explored bioresource around them

and have utilized as raw materials for food, medicine, clothes and other products

necessary for their life. In addition, bioresource has contributed to local development by

providing source of local specialties. For example, during 18-19 centuries

(Middle-Late EDO era), many feudal lords promoted to investigate and valorize their

local resources including bioresource to produce local specialties. This idea was

succeeded to the concept of “One-Village One Product” (OVOP), which was originated

from Oita prefecture of Japan and has been nowadays spread over the world. In this

context, bioprospection has a long history and has provided a way for local

development.

Recently, bioresource has been also recognized as a source of useful genes and

biological substances that can be applied to wide spectrum of industries such as food,

chemical, medicine, and cosmetics. Many organisms, especially plants and

microorganisms have been investigated to discover new genes that would produce

useful enzymes and molecules, or would enhance tolerance to biotic and/or abiotic

stresses. On the other hand, to find biological substances for new drugs,

pharmaceutical companies have been carrying out comprehensive screening on plant

species in tropical rainforest areas such as South America and Southeast Asia. As a

consequence, about one-forth of commercially available medicines are derived from the

molecules discovered from the bioresource in the tropical areas [1].

We are now carrying out bioprospecting research on North African region

especially Tunisia, which has the Mediterranean, semi-arid and arid environment. For

the maximum use of bioresources, we are keen to respect three “varieties”, variety of

biotope, variety of biological activities, and variety of usages [1].

So far bioprospection has been mainly focused on the tropical rainforest areas and

the temperate zones which are considered to have the richest variety of species, and

semi-arid and arid areas have been rarely explored. However, our bioprospecting

research on Tunisia revealed good potential of the bioresource in North African region

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for new useful bioactive substances. For example, the studies revealed that Tunisian

olive oils contain much more polyphenols than those of Europe [2], and we have

recently found novel antitumor and antimelanogenesis activities from Tunisian plants [3,

4]. The high potential of North African bioresource would come from the unique

geographical condition of the region where various climate characters from

Meditteranean area to Saharan desert appear in relatively small range (800 km from

north to south in Tunisia). In addition, harsh environmental stresses in semi-arid and

arid region such as strong sunshine, an appreciable change in temperature between

daytime and night, draught and salinity have stimulated the plant species to evolve

specific molecular mechanisms of tolerance. Thus through our research experiences,

we emphasize importance of exploring bioresource in various environmental conditions

(variety of biotope).

Recent bioprospection on useful genes and bioactive substances made targets of

screening more specific. However, screening for limited range of biological activities

may waste potential of bioresource. For example, screening for only antitumor

activity would pass up the chance to find novel other activities effective on other

diseases like allergy diabetes and Alzheimer’s disease. As shown in Fig. 1, many

medicinal effects have been detected and described on atopic dermatitis, allergy,

anti-oxidation, anti-pathogens and anti-inflammatory. On the other hand, however,

effects on pollen allergy, memory, hypercholesterolemia, immunity, protection of nerve

system and diuretic have rarely been reported. The bias of reported biological

activities can be attributed to lack of efficient screening method for the biological

effects, rather than lack of the effects themselves. Therefore it is very important to

establish various screening methods to explore bioresource for variety of biological

activities.

Screening and discovery of various biological activities gives a chance to a lot of

applications (variety of usages). Recently biological substances have been applied to

functional foods and cosmetics as well as medicines. In addition, the results of

bioprospecting research contribute to valorization of existing local specialties and to

their promotion to market. Especially the market of functional foods is becoming

bigger because of the trends of consumers concerned with health and natural products.

For example, dietary supplement including co-enzyme Q10 opened up a big market in

United States and Japan. In the case of North African products, studies on contents of

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polyphenols in Tunisian olive oil revealed their commercial advantage as functional

foods. In this context, we expect that our bioprospecting research will contribute to

local development of North African countries. In May 2007 in Tunis, Tunisia, we

co-organized a workshop on “Biotechnology for OVOP” to introduce our concept.

Fig. 1. Number of patents about medicinal activities derived from plants in

North African region, as of November 2005.

Again, we emphasize that success of bioprospection depends on whether one can

detect wide spectrum of useful activities and that therefore establishment of appropriate

screening systems is an important key point. So far, we have established more than 20

bioassay screening systems where biological activities are examined by responses of

cultured model mammalian cells (Fig. 2). Although bioassay has a disadvantage in

throughput comparing to the other in vitro screening methods, we still believe that it is

better method even for first-screening by some reasons. First, since bioassay is based

on the response of the human or other mammalian cells, the results will be expected to

reflect actual effects on individuals. Second we can also evaluate possible side effects

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of substances like cytotoxicity at the same time. Third, bioassay can be applied to

investigate molecular mechanisms of effects of biological substances on cells by

examining the cellular responses in detail. It will also help us for establishing better

screening systems.

In this textbook, we introduce protocols of several bioassay systems.

Considering convenience of their execution in laboratories in developing countries,

simplified protocols are also described. Our goal is to contribute to rural development

of North African and other developing countries through dissemination of bioassay

technology for bioprospecting to find materials for local specialty. We hope our

textbook will be a great help to it.

Fig. 2. Variety of biological activities which we can detect by our bioassay systems.

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References 1. Miyazaki, H., Isoda, H. Abe, Y and Nakamura, K. (2005) The 21st century-style of

bioprospecting research: overview and prospects of the project focused on arid land

bioresource. KAGAKU-TO-SEIBUTSU (Chemistry and Biology) 43, 482-485.

2. Abaza, L., Taamalli, W., Ben Temime, S., Ben Miled-Daoud, D., Gutiérrez, F. and M.

Zarrouk, M. (2005) Natural antioxidant composition as correlated to stability of

some Tunisian virgin olive oils. Rivista Italiana Delle Sostanza Grasse 82, 12-18.

3. Abaza. L,, Talorete, T.P., Yamada, P., Kurita, Y., Zarrouk, M., and Isoda, H. (2007)

Induction of Growth Inhibition and Dierentiation of Human Leukemia HL-60

Cells by a Tunisian Gerboui Olive Leaf Extract. Biosci Biotechnol Biochem., 71,

1306-1312.

4. Kawano, M., Matsuyama, K., Miyamae, Y., Shinmoto, H., Kchouk, M.E., Morio, T.,

Shigemori, H., and Isoda, H. (2007) Antimelanogenesis effect of Tunisian herb

Thymelaea hirsuta extract on B16 murine melanoma cells. Experimental

dermatology 16, 977-984.

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1. Human Intestinal Epithelium Model System

Caco-2 cells Considering the relevance of the food components and the effects of the oral route

for human exposure, we have investigated the TEER (Transepithelial electrical

resistance), permeability of LY (Lucifer yellow) and α-Glucosidase activity, by using

Caco-2 cell line, a well-known in vitro model of intestinal epithelium [1]. Caco-2 cells

are derived from a human colorectal carcinoma, with remarkable morphological and

biochemical similarity (tight junction, various enzyme, various transporter) to the

human small intestinal columnar epithelium [2].

The human adenocarcinoma cell line Caco-2 has been introduced as a model

system to study various aspects of intestinal biology [3]. Caco-2 cells form a

monolayer of polarized cells when grown on a permeable support in a two-chamber

system and after confluency spontaneously develop a number of features characteristic

for differentiated enterocytes including the expression of brush border enzymes such as

lactase (cleaves milk sugar), sucrase-isomaltase (cleaves cane sugar and breakdown

products of starch and glycogen), and dipeptidylpeptidase [4].

Therefore, this study also investigated how efficiently sample infusion could

inhibit the carbohydrate digestion by measuring the liberated extracellular glucose

concentration on the apical and basal sides of Caco-2 cell monolayers.

[Caco-2 cells]

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Cell Culture of Caco-2 Caco-2 cells (obtained from ATCC) were maintained routinely in DMEM

(Dulbeco´s Modified Eagle´s Medium) supplemented with 10 % fetal bovine serum

(FBS). Cells were grown to 80-90 % confluency prior to setting up the drug transport

experiment in the transwell plates (CORNING) [5].

Protocol 1

Measurement of TEER (Transepithelial electrical resistance) Monolayers integrities, thus their barrier function and permeability function, were

determined either by measuring the transepithelial electrical resistance (TEER).

Caco-2 monolayer formation was assessed by measuring TEER using Millipore

Millicell-ERS device (Cat # MERS 000 01) and a World Precision Instruments probe

(WPI, Cat # STX 100M). The transwell plates were then washed three times with

HBSS (Hank's Buffered Salt Solution) buffer, pH 7.4 (Invitrogen), incubated for half

hour at 37oC [6, 7].

[Measurement of TEER]

MATERIALS

Millipore Millicell-ERS device (Cat # MERS 000 01)

World Precision Instruments probe (WPI, Cat # STX 100M)

HBSS (Hank's Buffered Salt Solution) buffer, pH 7.4 (Invitrogen)

METHOD

1. Caco-2 cells were seeded at a density of 1×105 cells/cm2 and a monolayer was

formed after culturing at 37oC, 5% CO2 for 10 or 21 days.

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2. Caco-2 cells were fed apically and basolaterally with fresh medium every other

day.

3. The integrity of the cell layer was evaluated by measurement of transepithelial

electrical resistance (TEER) with Millicell-ERS equipment.

4. The monolayer of cells was gently rinsed twice with HBSS and left to equilibrate

in the same solution for 30min at 37°C.

5. A monolayer with a TEER of more than 300 Ω cm2 was used for the

transepithelial transport experiments.

Protocol 2

Measurement of LY (Lucifer yellow) Monolayers integrities, thus their barrier function and permeability function, were

determined either by measuring the lucifer yellow (LY), that leaks from the apical

chamber between the cells down into the basolateral chamber.

[Measurement of LY]

MATERIALS

Lucifer yellow (LY) (Sigma)

HBSS

METHOD

1. Caco-2 cells were seeded at a density of 1×105 cells/cm2 and a monolayer was

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formed after culturing at 37oC, 5% CO2 for 10 or 21 days.

2. Caco-2 cells were fed apically and basolaterally with fresh medium every other

day.

3. Transwell plates were disassembled and the Caco-2 monolayer integrity was

measured by adding 75µL 100µg/mL of lucifer yellow (LY) (Sigma) in HBSS

buffer, pH 7.4 to each filter well. 250µL of HBSS buffer, pH 7.4 was added to the

bottom wells.

4. The plates were reassembled and then incubated for 2 hours with shaking (60 rpm)

at room temperature. Lucifer yellow fluorescence (RFU) was measured at

485/535nm [8].

Percent rejection of LY was calculated using the equation:

Protocol 3

Measurement of αααα-Glucosidase activity α-Glucosidase enzymes in the intestinal lumen and in the brush border membrane

play main roles in carbohydrate digestion to degrade starch and oligosaccharides to

monosaccharides before they can be absorbed. It was proposed that suppression of the

activity of such digestive enzymes would delay the degradation of starch and

oligosaccharides, which would in turn cause a decrease in the absorption of glucose and

consequently the reduction of postprandial blood glucose level elevation [9].

MATERIALS

Phosphate-buffered saline (PBS)

28 mM sucrose solution

28 mM maltose solution

Glucose CⅡ test kit (WAKO)

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[ Glucose oxidase method ]

METHOD

1. Caco-2 cells were fed on polyethylene terephthalate membranes (Falcon, pore

size: 0.4 μm, pore density 1.6×106 pores/cm2, diameter: 23.1 mm) in a 6 well

plate.

2. After cells reached 100% confluence (5 days), cells were grown for 14 to 20 days

more.

3. Then, cell culture medium was removed and both the apical and basal chambers

were washed 3 times with 2 ml of phosphate-buffered saline (PBS).

4. The culture medium in the apical chamber was replacedwith a reaction mixture

consisting of sample (0.2 ml) and 28 mM sucrose solution in PBS (0.8 ml) and 28

mM maltose solution in PBS (0.8 ml) as substrates for sucrase and maltase

inhibitory activity determination, respectively.

5. In the basal chamber 1 ml of PBS was added instead of the culture medium.

6. The assay plate was then incubated at 37 °C in 5% CO2 atmosphere for 3 h.

7. After incubation the liberated extracellular glucose concentration in the cell free

solution of both apical and basal chamber was measured by the glucose oxidase

method using the glucose CⅡ test kit (WAKO) [10].

******* To confirm the applicability of this model system and compare the inhibitory

effect of mulberry tea to commercial α-glucosidase inhibitor, 1-deoxynojirimycin,

known as a potent medicine for diabetes, was used as a positive control.*************

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Reference 1. Y. Sambuy, I. De Angelis, G. Ranaldi, M.L. Scarino, A. Stammati and F. Zucco, The

Caco-2 cell line as a model of the intestinal barrier: influence of cell and

culture-related factors on Caco-2 cell functional characteristics, Cell Biology and

Toxicology 2005, 21, 1–26.

2. Asano, N.; Yamashita, T.; Yasuda, K.; Ikeda, K.; Kizu, H.; Kameda, Y.; Kato, A.;

Nash, R. J.; Lee, H. S.; Ryu, K. S., Polyhydroxylated Alkaloids Isolated from

Mulberry Trees (Morus alba L.) and Silkworms (Bombyx mori L.), J Agric Food

Chem 2001;49, 4208-4213.

3. Zweibaum, A., Laburthe, M., Grasset, E. and Louvard, D. (1991). The use of

cultured cell lines in studies of intestinal cell differentiation and function. In:

Handbook of Physiology (Field, A. and Frizzel, R.A., eds.), Amer. Physiol. Soc.

Bethesda. 4, 223-255.

4. Louvard, D., Kedinger, M., and Hauri, H.-P. (1992). The differentiating intestinal

epithelial cell: Establishment and maintenance of functions through interactions

between cellular structures. Ann. Rev. Cell Biol. 8, 157-195.

5. Isoda, H., Han, J., Tominaga, M., Maekawa, T., Effect of capsaicin on human

intestinal cell line Caco-2, Cytotechnology, 2001, 36, 155-161.

6. Han, J., Isoda, H., Maekawa, T., Analysis of the mechanism of the tight-junctional

permeability increase by capsaicin treatment on the intestinal Caco-2 cells,

Cytotechnology, 2002, 40, 93-98.

7. Han, J., Akutsu, M., Talorete, T. P. N., Maekawa, T., Tanaka, T., Isoda, H.,

Capsaicin-enhanced ribosomal protein P2 expression in human intestinal Caco-2

cells, Cytotechnology, 2005, 47, 89-96.

8. Y. Konishi, S. Kobayashi and M. Shimizu, Tea polyphenols inhibit the transport of

dietary phenolic acids mediated by the monocarboxylic acid transporter (MCT) in

intestinal Caco-2 cell monolayers, J. Agric. Food Chem. 2003, 51, 7296–7302.

9. Takuya Suzuki and Hiroshi Hara, Difructose anhydride III and sodium caprate

activate paracellular transport via different intracellular events in Caco-2 cells, Life

Sciences, 79, 2006, 401-410.

10. Jongwon Park, Chang-Soo Kim, Shanrui Zhang and Minsu Choi, Glucose Oxidase

(GOD)-Coupled Amperometric Microsensor with Integrated Electrochemical

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Actuation System, Instrumentation and Measurement Technology Conference,

Ottawa, Canada, May 2005, 17-19.

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2. Human Leukemia Cells Differentiation Model

System

Introduction Cell differentiation is essential for normal growth and homeostasis. Some cancer

cells including leukemia cells exhibit a defect in their capacity to mature to

nonreplicating adult cells, thereby remaining in a highly proliferative state and

outgrowing their normal cellular counterparts. The induction of terminal

differentiation, leading to the eventual elimination of tumorigenic cells and reestablish

normal cellular homeostasis, represents an alternative approach to the treatment of

cancer by conventional antineoplastic agents.

Some of the compounds, including vitamin A, have shown limited success in the

prevention of cancer and anti-cancer therapy. The mechanism of action of the

substances in chemoprevention and therapy of cancers involves modulation of cell

proliferation and differentiation. The human promyelocytic leukemia HL-60 cell line

is considered an excellent model for studying cellular differentiation in vitro [1].

HL-60 cells HL-60 cells have been induced to differentiation into mature granulocytes

[2-3] and macrophage-like cells [4-5] and consists of stem-like cells that are bipo-tent

with respect to myeloid or macrophage differentiation [6]. HL-60 cell line is a

promyelocyte which proliferates continuously and is known to differentiate into

granulocytes or monocytes by variety of compounds, such as butylate, dimethyl

sulfoxide, and vitamin D3 [2, 7]. Treating HL-60 cells with 1, 25-dihydroxyvitamin

D3 (1,24-(OH)2D3) or all-trans retinoic acid causes them to differentiate into

monocytes or granulocytes, respectively These compounds, among others, are used

as chemotherapeutic agents for acute promyelocytic leukemia (APL); however, effective

doses of these drugs also produce objectionable side effects, physiological toxicity and

drug resistance [2, 8-9].

As an alternative approach to APL treatment, the induction of differentiation of

leukemia cells is favored because cells exposed to chemical or biological inducers of

differentiation do not undergo the cytodestruction associated with cytotoxic agents;

instead, they acquire the phenotypic characteristics of end-stage adult cell forms and

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undergo programmed cell death [10]. However, there is a need to identify nontoxic

differentiation inducers, particularly from natural and food sources, to be used not only

as safe chemotherapeutic agents but for prophylactic purposes as well.

Here are shown four assays, which are useful for screening of substances that

differentiation in the HL-60 cells. Therefore, the method of searching for the revulsive

is shown here.

Protocol 1

1. Cell proliferation assay 1-1. Cell Culture

Cells

HL-60 cells (Riken Cell Bank, Tsukuba, Ibaraki, Japan).

Medium

Phenol-red-free RPMI 1640 medium (Gibco).

Fetal bovine serum (FBS, Sigma).

Penicillin - streptomycin solution (ICN Biomedicals Inc.).

Special Equipment

Cleanbench.

CO2-incubator.

75 cm2 tissue culture plate.

Phase contrast microscopy.

HL-60 cells are grown in phenol-red-free RPMI 1640 medium supplemented with

10% heat-inactivated fetal bovine serum and 1% penicillin (50.0 mg/ml) - streptomycin

(50.0 ml/ml) solution at 37oC in a 5%CO2 incubator. The medium is replaced every

two days after checking cell growth under the microscope.

1-2. MTT assay MATERIALS

96 well microplate (Falcon).

Microplate reader.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide.

(MTT) (Dojindo).

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Sodium Dodecyl Sulfate (SDS, Sigma).

METHOD

The MTT assay is a sensitive and quantitative colorimetric assay that is used to

determine cell viability and proliferation [11].

1. At approximately 60 - 80% confluence, HL-60 cells were harvested and seeded

onto 96-well plates at 4 x 104 cells per well in 50 µl of medium.

2. After overnight incubation (at least 12 h), add 50 µl samples with medium to

obtain the required final concentration.

3. After 48 h incubation, add 10 µl of 5 mg/ml MTT.

4. After 24 h incubation, add 100 µl of 10% SDS.

5. After 24 h incubation to completely dissolve the formazan produced by the cells.

The absorbance was then spectrophotometrically determined at 570 nm using a

multidetection microplate reader.

6. Blanks are prepared at the same time to correct for the absorbance caused by

sample color and by the inherent ability of a sample to reduce MTT in the absence

of cells.

7. The optical density of the formazan produced by the untreated control cells is

considered as representing 100% viability.

Protocol 2

DNA fragmentation assay MATERIALS

Buffer and solution

Ca2+- and Mg2+-free phosphate-buffered saline (PBS-)

pH 7.4; PBS; 20 mM sodium phosphate and 130 mM NaCl

DNA Extractor WB Kit (Sodium Iodide Method - Wako) or DNA Purification Kit

(Wizard® Genomic DNA, Promega)

TE buffer

10 mmol/l Tris-HCl (pH8.0), 1 mmol/l EDTA (pH8.0)

Loading buffer (Wako)

0.02% Bromophenol blue

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0.02% Xylene cyanol FF

50% Glycerol

1% SDS

Ethidium bromide (Wako).

Molecular weight marker (Marker 1 (l/Hind III digest) Wako).

Special Equipment

Microplate reader.

METHOD

1. Inoculate Petri plates with 1.0 x 106 cells per plate in 10 ml of medium.

2. Incubate overnight.

3. Add samples to obtain the desired final concentrations.

4. Incubate the cells for a minimum of 4 h.

5. Use the DNA Extractor WB Kit or the DNA Purification Kit for the DNA

extraction.

6. Determine the DNA amount by measuring the optical density of the sample at 260

and 280 nm. If necessary, dilute the DNA obtained in 100 ml of TE buffer.

7. The amount of DNA to be loaded into each well should be 1 µg. Therefore,

depending on the DNA concentration, 2-10 ml of the DNA sample is mixed

thoroughly with 2 ml of loading buffer.

8. This mixture is then loaded into the wells of a 2% electrophoresis-grade agarose

gel containing 0.5 mg/ml ethidium bromide (EtBr). If the ethidium bromide is not

added directly to the gel, the gel may be stained for 10 min in a solution

containing 10 µg/ml EtBr in TE buffer.

9. Electrophoresis is carried out for 25-30 min at 100 V with a molecular weight

marker.

Protocol 3

Cell differentiation assays (NBT assay) HL-60 differentiation was assessed using the NBT assay and the double

staining esterase assay. The NBT reduction ability is determined by the method of

Takeda et al. [12].

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MATERIALS

Nitroblue tetrazolium chloride (NBT, Wako).

24-well plates (Farcon)

METHOD

1. HL-60 cells (2.5 x 106 cells in 10 ml of medium) were incubated overnight in Petri

plates in the presence of sample.

2. The cells were harvested, adjusted to 2.0 x 105cells/ml and resuspended in culture

medium with 0.1% NBT.

3. The obtained cell suspensions were seeded in 24-well plates and incubated at 37oC

for 2 - 3 h.

4. The percentage of cells containing blue-black formazan deposits is determined by

counting at least 200 cells under phase contrast microscopy.

Protocol 4

Cell differentiation assays (Double staining esterase assay) To identify specific and nonspecific esterase activities, use a commercially

available kit.

MATERIALS

8-well Lab Tek® chamber slides (Nalge Nunc International, Apolgent Technologie

Company, USA).

NAFTOL AS-D KLORACETAT-ESTERASE OG,

α-NAPHTHYLACETATESTERASE (Sigma-Aldrich, St. Louis, MO).

METHOD

1. For the double staining esterase assay, HL-60 cells (6.0 x 104 cells per well in 300

µl of medium) were seeded in 8-well Lab Tek® chamber slides.

2. After overnight incubation, add samples to obtain the desired final concentrations

3. Incubated for 24 h.

4. Submerge slides on fixer solution for fixation during 30 seconds.

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Mix vigorously the last 5 seconds

Use α-Naphtyl acetate esterase (NAE) and naphtol As-D chloroacetate esterase

(NCAE) to detect monocytes (black granulation) and granulocytes (red

granulation).

5. Rinse with 200 µl deionized water (Mili Q).

6. Add 200 µl Green solution.

7. Incubate 30 min at 37 shelter from light.

8. Wash with 200 µl Mili Q during 5 min.

9. Add 200 µl Red solution.

10. Incubate 15 min at 37 shelter from light.

11. Rinse with 200 µl Mili Q at least during 2 min.

12. Add 200 µl hematoxylin (fixing) solution for 2 min.

13. Wash at tap water then dry at room temperature.

14. Examine under microscope

Preparation of the fixer solution

① 25ml of citrate solution

② Add 6ml acetone

③ Add 8ml 37% formaldehyde

Notice: to store in a refrigerator until use.

Preparation of the α-Naphtyl acetate esterase procedure (Green solution)

Warm some deionized water for using substate at 37.)

① Put 1ml Fast blue BB. on flask

② Add 1ml of sodium nitrite and mix gently and replace 2 min avoiding gas

bubbles.

①＋②＝Solution A (The color pass from brown to yellow)

③ Add 40ml of warm Mili Q to solution A.

④ Add 5ml TRIZMAL 7,6 concentrated tampon.

⑤ Add 1ml of α-Naphtyl acetate esterase solution

Preparation of the Naphtol As-D chloroacetate esterase procedure (Red solution)

Warm some Mili Q for using substate at 37.

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① Put 1ml Fast Red Violet LB. on flask.

② Add 1ml of sodium nitrite (NaNO2) and mix gently and replace 2 min

avoiding gas bubbles

①＋②＝Solution B

③ Add 40ml (4ml) of warm Mili Q to solution red solution

④ Add 5ml TRIZMAL 6,3 concentrated tampon.

⑤ Add 1ml of Naphtol As-D chloroacetate esterase solution.

Reference 1. Kang, S. N., Chung, S. W., and Kim, T.S., Capsaicin potentiates 1,25-

dihydroxyvitamin D3- and all-trans retinoic acid-induced differentiation of human

promyelocytic leukemia HL-60 cells. Eur. J. Pharmacol., 420, 83-90 (2001).

2. Breitman, T.R., Selonick, S.E., and Collins, S.J., Induction of differentiation of the

human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl.

Acad. Sci. U.S.A., 77, 2936-2940 (1980).

3. McCarthy, D. M., San Miguel, F., Freake, H. C., Green, P. M., Zola, H., Catovsky,

D., and Goldman, J. M., 1, 25-Dihydroxyvitamin D3 inhibits ploliferation oh human

promyelocytic leukemia (HL-60) cells and induces monocyte-macrophage

differentiation in HL-60 and normal human bone marrow cells, Leuk. Res. 7, 51-55

(1983).

4. Collins, S. J., Gallo, R. C., and Gallagher, R. E., Continuous Growth and

differentiation of human myeloid leukemia cells in suspension culture, Nature, 270,

347-349 (1977).

5. Rovera, G., Santoli, D., and Damsky, C., Human promyelocytic leukemia cells in

culture differentiate into macrophage–like cells when treated with a phorbol diester,

Proc. Natl. Acad, Sci. USA, 76, 2779-2783 (1979).

6. Fortana, J. A., Colbert, D. A., and Deisseroth, A. B., Identification of a population of

biopotent stem cells in the HL-60 human promyelocytic leukemia cell line, Proc.

Natl. Acad, Sci. USA, 78, 3863-3866 (1981).

7. Tanaka, H., Abe, E., Miyaura, C., Shiina, Y., and Suda, T., 1α, 25-dihydroxyvitamin

D3 induces differentiation of human promyelocytic leukemia cells (HL-60) into

monocytic macrophage, but not into granulocytes. Biochem. Biophys. Res.

Commun., 117, 86-92 (1983).

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8. Warrell Jr., R.P., Frankel, S.R., Miller Jr., W.H., Scheinberg, D.A., Itri, L.M.,

Hittelman, W.N., Vyas, R., Andreeff, M., Tafuri, A., and Jakubowski, A.,

Differentiation therapy of acute promyelocytic leukemia with tretinoin

(all-trans-retinoic acid). N. Engl. J. Med., 324, 1385-1393 (1991).

9. Frankel, S.R., Eardley, A., Lauwers, G., Weiss, M., and Warrell Jr., R.P., The

“retinoic acid syndrome” in acute promyelocytic leukemia. Ann. Intern. Med., 117,

292-296 (1992).

10. Beere, H.M., and Hickman, J.A., Differentiation: a suitable strategy for cancer

chemotherapy? Anti-Cancer Drug Des., 8, 299-322 (1993).

11. Mossman, T., Rapid colorimetric assay for cellular growth and survival: application

to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55−63 (1983).

12. Takeda, K., Hosoi, T., Noda, T., Arimura, H., and Konno K., Effect of

fibroblast-derived differentiation inducing factor on the differentiation of human

monocytoid and myeloid leukemia cell lines. Biochem. Biophys. Res. Commun.,

155, 24-31 (1988).

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3. Assay Systems for Screening of Substances that

Protect the Liver from Hepatic Injuries

Introduction The liver is an organ localized as a guardian interposed between the digestive tract

and the rest of the body. The major function of the liver involves effective uptake of

substrates from the intestine and their subsequent storage, metabolism, and distribution

to blood and bile. Another function of the liver is biotransformation of exogenous

xenobiotic pollutants, drugs, and endogenous metabolites.

The structural components of the liver include plates of parenchymal hepatocytes

separated by vascular channels known as sinusoids. Hepatic stellate cells (also called as

vitamin A-storing cells, lipocytes, fat-storing cells and Ito cells) reside in the space

between parenchymal cells and sinusoidal endothelial cells of the hepatic lobule.

Here are shown two assays, which are useful for screening of substances that

protect lipid peroxidation in the hepatocytes or inhibit activation of hepatic stellate cells.

Protocol 1 1. Lipid peroxidation assay using mouse hepatocytes

Loosely bound iron, not transferrin-bound iron, can work as a catalyst for free

radical-mediated oxidative tissue damage. Such iron supplied by treatment with ferric

nitrilotriacetate (Fe-NTA) induces the lipid peroxidation in the hepatocytes. The degree

of lipid peroxidation in the hapatocytes by Fe-NTA treatment was accessed by the

thiobarbituric acid-reactive substances (TBARS) in the culture supernatant of

hepatocytes.

1-1. Preparation of mouse hepatocytes METHOD

1. Incubate pre-perfusion solution (SC-1) and perfusion solution (SC-2) containing

0.5 mg /ml of collagenase in the water bath at 38~40°C. (“Supply these solutions

at 37°C in the tissue”.) Prepare each solution as follows.

22

2. Connect two sterilized extension tubes (Top, X1-50) to both ends of a sterilized

silicon tube, which is equipped in a Perista pump.

3. Fill the tubes with Pre-perfusion solution (SC-1) using the pump.

4. Anesthetize a mouse by injecting pentbarbital intraperitoneally.

5. Make a midline incision and push the intestinal content aside everting them to

expose portal vessels.

6. Insert a 24-gauge plastic needle with a 27-gauge needle (Terumo SR-OT2419CW)

into portal vein. After removing the inside needle, the plastic needle in the portal

vein is fixed with a thread. Blood will flow to the needle.

7. Connect the extension tube filled with SC-1 solutions (“Do not perfuse air bubbles

to the liver”) and perfuse SC-1 solution at the flow rate of 6 ml / min for 7 min. At

the beginning of the perfusion, cut the inferior vena cava to flow out the perfused

solutions. Complete tissue bleaching; a light tan color indicates adequate

perfusion.

8. Stop the pump and change the solution to collagenase solution (0.5 mg/ ml

collagenase in SC-2). Perfuse the collagenase solution at the same flow rate for ~

10 min using the pump.

9. Excise the perfused liver, placing it in a 100-mm plastic Petri dish and cut with a

blade and a forceps. Add 10 ml of serum free William’s E medium and disperse

the cells with very gentle pipetting using a pipette having a wide top.

10. Transfer the liver cells into a 50 ml plastic tube. Add 30 ml of serum free

William’s E medium and suspended gently using a pipette having a wide top.

11. Pass the liver cell susupension through a 70 mm mesh.

12. The liver cell susupension is centrifuged at 50 x g for 1 min at 4°C.

13. Remove the supernatant and add 10 ml of William’s E medium containing 10%

FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 1% glutamine

(serum-containing William’s E medium) and gently suspended the liver cells with

very gentle pipetting.

14. Centrifuge at 50 x g for 1 min at 4°C. Repeat the procedure 11~13 for 4 times to

remove the non-parenchymal cells.

15. A final cell pellet consists of mostly parenchymal hepatocytes is suspended with

23

10 ml of serum-containing William’s E medium.

16. Count viable cells (hepatocytes) using trypan-blue staining.

1-2. Lipid peroxidation assay 1-2-1. Lipid peroxidation by the treatment of Fe-NTA in hapatocytes.

1. Inoculate 106 hepatocytes onto a collagen-coated 6 well plate. Culture the cells

in 2 ml of serum-containing William’s E medium at 37°C in a 5%CO2

atomosphere and 100% humidity.

2. After 4 h, remove the cell medium and induce lipid peroxidation in hepatocytes by

incubating the cells for 5 h in 0.8 ml of serum-free William’s E medium with 100

mM ferric-nitrilotriacetate solution (Fe-NTA) in the presence or absence of

substances that may have anti-lipid peroxidation activity. (This system represents

a well-established method to induce lipid perxidation both in vivo and in vitro,

and it has been shown that approximately 95 % of the total MDA is released into

the culture medium within 5 h. (Shimizu et al. (1999))

Reference Shimizu, I., Ma, Y-R., Mizobuchi, Y., Liu, F., Miura, T., Nakai, Y., Yasuda, M.,

Shibata, M., Horie, T., Amagaya, S., Kawada, N., Hori, H., and Ito, S. 1999,

Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats.

Hepatology 29: 149.

Preparation of Fe-NTA solution

1. Prepare a solution of 80 mM ferric nitrate solution in water.

2. Prepare a solution of 160 mM nitrilotriacetic acid disodium salt in water.

3. Mix these solutions in a volume ratio of 1:2 and adjust the pH of the solution

with sodium hydrogen carbonate (conc. Fe-NTA solution).

4. Dilute the conc. Fe-NTA solution with serum free William’s E medium to

prepare 100 mM Fe-NTA solution in each assay.

5. Recover the culture supernatant, centrifuged it at 500 x g for 5 min at 4°C and

recover the supernatant for lipid peroxidation assay.

24

I-2-2. Lipid peroxidation assay (A very simple method)

1. Add 100 ml of 2.8% Trichloroacetate solution and 100 ml of 1% thiobarbituric

acid solution in 0.05N NaOH to 40 ml of the culture supernatant of the

hepatocytes in a tube with a screw cap.

As a positive control, 40 ml of 20 mM 1,1,3,3 tetramethoxypropane solution is

used.

2. Boil the solution in a tightly closed screw-capped tube for 10 min at 100 °C.

The solution containing peroxidized lipid substances turn red.

3. Keep the solution on ice for 10 min.

4. Centrifuged at 12,000 rpm for 10 min at 4°C.

5. Transfer the 100 ml of the supernatant to a 96 well plate.

6. Measure absorbance at 532 nm of the supernatant by a microplate reader.

Note. If you measure the lipid peroxidation precisely, please refer the following papers.

Reference Kosugi, H., and Kikugawa, K. 1989. Potential thiobarbituric acid-reactive

substances in peroxided lipids. Free Radic. Biol. Med. 7: 205

Kikugawa, K., Yasuhara, Y., Ando, K., Koyama, K., Hiramoto,K., and Suzuki, M.

2003. Protective effect of supplementation of fish oil with high n-3 polyunsaturated

fatty acids against oxidative stress-induced DNA damage of rat liver in vivo. J.

Agric. Food Chem. 51: 6073.

2. Anti-fibrosis assay by the use of a hepatic stellate cell line. The hepatic stellate cells are responsible for the majority of extracellular matrix

deposition in the normal and fibrotic liver. In the normal liver, hepatic stellate cells

regulates the vitamin A homeostasis and stored it as retinyl palmitate in the lipid

droplets in the cytoplasm. In the chronic injury, the hepatic stellate cells are activated

and transdifferentiate to myofibroblastic cells and produce extensively extracellular

matrix, such as collagen type I and fibrosis is developed. TGF-b produced by the

Kupffer cells or activated hepatic stellate cells functions as a fibrosis promoting factor.

Fibrosis was once thought to be irreversible, however, recent studies suggested

25

that fibrosis, to some extent, is reversible when the underlying source of liver injury is

treated. Production of collagen, one of the markers for activation of hepatic stellate cells,

can be monitored by Sirius Red staining method (Walsh et al. (1992)) to examine the

inhibition of stellate cell activation by exogenous factors.

Reference

Walsh, B. J., Thornton, S. C., Penny, R., and Breit, S. N. 1992. Microplate

reader-based quantification of collagens. Anal. Biochem. 203; 187

2-1. Sirius Red assay to quantify the collagens 1. Inoculate 0.5 ml of 5 x 104 cells / ml of HSC-T6 cells (generously provided by

Prof. Scott L. Friedman) in Dulbecco’s modified Eagle (DME) medium

containing 10 % FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in a

24-well plate.

2. Culture the cells at 37°C in a 5% CO2 atomosphere and 100% humidity for 24 h.

3. Remove the medium and wash the cells with 0.5 ml of PBS.

4. Add 0.5 ml of serum free DME medium with or without 1 ng/ ml TGF-b.



7. Add 0.5 ml of serum free DME medium with or without a test sample which is

considered to be included anti-fibrosis activity.



10. Add 0.5 ml of 0.5 M acetic acid and keep the plate at room temperature for 30

min.

11. Add 0.25 ml of 0.1% Sirius Red F3B (Avocado Research Chemicals, Direct red

80) in saturated aqueous picric acid solution and keep the plate at room

temperature for 2 h.

12. Remove the solution and wash with 0.5 ml of 10 mM HCl 5 times.

13. Add 0.25 ml of 0.1M NaOH and keep the plate at room temperature for 5 min.

14. Transfer 200 ml of the solution to a 96-well plate.

15. Measure absorbance at 540 nm by a microplate reader.

26

Note. If you would like to use HSC-T6 cells, please contact Professor Scott L. Friedman,

Mount Sinai School of Medicine, USA.

Reference Vogel, S., Piantedosi, R., Frank, J., Lalazar, A., Rockey, D. C., Friedman, S, L,,

and Blaner. W. S. 2000. An immortalized rat liver stellate cell line (HSC-T6): a

new cell model for the study of retinoid metabolism in vitro. J. Lipid Res. 41;

882.

27

4. Production of Mouse Monoclonal Antibody

Introduction Immunological methods, such as Enzyme linked immunosorbent assay, flow

cytometry, Western blotting, immunochromatography, are widely used to detect

biological molecules such as proteins, sugar chains, and low molecular weight haptens.

Sera from animals immunized with certain antigens include polyclonal antibodies.

Those polyclonal antibodies can recognize different antigen binding site (epitopes) of

antigens.

Monoclonal antibody is an antibody secreted by single cloned antibody-producing

cell. Each cloned cell secrets antibody against single epitope. Such antibody is very

useful to detect certain epitope with high affinity. In this article, classical method to

obtain a hybridoma secreting mouse monoclonal antibody is described.

Outline of the methods*

28

1. Immunize mice with an antigen (usually proteins)

2. Prepare spleen cells and fuse with mouse myeloma cells

3. Select fused cells (hybridomas) with HAT-medium

4. Analyze antibody-secretion

5. Clone antibody-positive hybridoma

6. Culture the hybridoma to obtain monoclonal antibody of interest

* Basic cell culture and animal techniques are required.

Protocol 1

Immunize Balb/c mice with your antigen 1. Antigen: Purified antigen protein is preferable. While some articles said

there is no need to purify your protein, it would be better using purified protein

as an antigen. Sometime you will see monoclonal antibodies to proteins

contaminated in your antigen.

Dissolve your protein into sterile saline or phosphate buffered saline (up to a

protein concentration of 0.5 mg/ml). Two 1ml-glass syringes with 0.5 ml

antigen solution and 0.5 ml Freund Complete Adjuvant are connected and

emulsified with pumping syringes. Plastic syringes are not preferable. To

check the condition of the resulted emulsion, drop the emulsion onto water

surface. The stable emulsion flow in the water keeping it’s shape.

2. Immunization: Inject 0.5 ml adjuvant-emulsified antigen to Balb/c mice (6-8

week old, female of male) i.p. Two to three weeks later, inject antigen without

adjuvant i.v. or i.p. If you have any antibody checking method, collect small

amount of blood from mice and measure antibody titers

1. Preparation of myeloma cells

1. Cell line: Mouse myeloma SP2/O (SP2/O-Ag14) does not produce any

immunoglobulin poly peptide. Other myeloma NS-1 or P3X63 would also be

available. Maintain nyeloma cells with Dulbecco modified Eagle medium

(DMEM) supplemented with fetal calf serum (FCS, also known as fetal bovine

29

serum FBS).

2. Culturing myeloma cells: Maintain myeloma cells least twice a week by

splitting ratio of 1:10 to 1:20. Because SP2/O cells lightly adhere on a culture

dish, strong pipetting to the culture dish surface is required to suspend cells.

Add 10ml of flesh FCS-DMEM to 0.5 ml of suspended cells and culture at 37 C

(5 % CO2, 95 % air, CO2 incubator). Three days before cell fusion, prepare ten

10 cm dishes with 1:20 split ratio. The day of cell fusion, harvest SP2/O

myeloma cells carefully into 50 ml culture tube and centrifuged (1,200 rpm for

10 min). Remove supernatant and re-suspend cells with DMEM (without FCS).

Centrifuge cells and continue washing procedure for three times. Finally,

suspend into DMEM (without FCS) and count cell density.

2. Preparation of mouse spleen cells 1. Spleen: Lightly anesthetized mouse is sacrificed and collect a spleen

acseptically into 6 cm sterile plastic dish. Spleen is a red-back organ placed

near liver. Remove adipose tissue around the spleen.

2. Spleen cell suspension: Prior to cell fusion, spleen cells (splenocytes) should

be suspended. Add 10 ml DMED into 10 cm dish and put collected spleen

carefully. Push spleen between sterile slide glasses to make spleen crushed.

Spleen is a suck and spleen cells are easily obtained crushing spleen suck.

Suspend spleen cells with vigorous pipetting and move to 50 ml culture tube.

Wash the dish with DMEM and combine to the culture tube and suspend cells

vigorously. Allow to stand few minutes to precipitate large tissue debris and

upper part of the medium is collected to another culture tube. Centrifuge the

tube and wash the spleen cells for three times. Re-suspend the cells and count

spleen cells. Spleen cells contain red blood cells. Be careful not cont red

blood cells as lymphocytes.

3. Cell fusion and HAT selection culture 1. Day 0; Cell fusion: Mix 5x10^7 SP2/O myeloma cells and 5x10^7 spleen

cells and mix the cells. Centrifuge the tube at 1,500 rpm for 15 min (without

30

breaking centrifuge). Remove supernatant.

Add 1ml pre-warmed (37 C) 50 % polyethylene glycol (PEG) very slowly (1

ml/mil) with gentle pipetting. Rotate the tube with heating on your palm for 2

min and the tube is allowed to stand for 5 min. Add 1 ml pre-warmed DMEM

and mix gently to dilute PEG solution. Add 2, 4, and 8 ml of DMEM each 30

second.

Centrifuge the tube at 1,000 rpm for 15 min and precipitated cells are

resuspended into 70 ml of pre-warmed FCS-DMEM (use a 100ml sterile

medium bottle). Pipet 140 ul of well-suspended cell mixture into five 96-well

microculture plates and culture the plate in CO2 incubator.

2. Day 1-; HAT selection: Prepare FCS and HAT supplemented DMEM. In

this medium, A (aminopterine) kills HGPRT deficient SP2/O myeloma cells and

spleen cells can not grow usually. So, the cells grow HAT supplemented

medium are fused hybridoma cells.

Next day of the fusion (Day 1), add 100 ul of HAT medium. On the Day 2,

remove half amount of the culture medium and add flesh HAT medium. This

procedure is repeated twice a week for two to four weeks. During the culture,

large hybridoma cells are observed under microscopic observation. Please be

careful not to allow over growth of hybridomas.

4. Enzyme Linked Immunosorbent Assay (ELISA) 1. Preparation of antigen coated plate: Dilute antigen protein (0.1-10 ug/ml) with

0.05 mol/l sodium bi carbonate and pipet 60 ul into the wells of 96 well micro

ELISA plate. Adhere the protein onto the plate at 4 C for overnight.

Discard the antigen solution and to prevent non-specific antibody binding, block

the wells with Block Ace (1/4-1/5 diluted) or 2% Bovine Serum Albumin at

room temperature for 1 h. Wash the plates with phosphate buffered saline

(PBS) containing 0.05% Tween-20 (PBS-Tween).

2. Reacting culture supernatant on the well: 50uL of the culture supernatants are

pipetted into blocked ELISA plates and incubated at room temperature for 1 h.

Wash the plate for three times with PBS-Tween. Add 1/1,000 –

31

1/10,000diluted anti mouse IgG conjugated with HRPO and incubate at room

temperature for 1 h. Wash the plate with PBS-Tween for six times before

adding substrate solution.

Add 100 ul of substrate solution to each well of ELISA plate and the color

developed is measured with plate reader of take digital photos of the plates.

3. Confirmation of specificity of the antibodies: Sometimes non-specific or

blocking reagent specific antibodies are obtained. To eliminate this, antibody

positive supernatants are subjected ELISA without antigen protein. To confirm

the specificity, Western blot analysis with monoclonal antibody obtained is

required.

5. Cell cloning of hybridoma cells 1. Oligo-clonal expansion: Before cloning, hybridoma cells in antibody positive

wells are first transferred to 24 well culture plate, and next to 6cm dish.

Change medium without HAT and store cells in frozen (at -80 C).

2. Cell cloning: Plate 100 hybridoma cells into three to five 96 well microculture

plates and culture cells for 2 to 4 weeks. When the clones become visible,

antibody in the supernatant is measured by ELISA. Expand the

antibody-positive hybridoma cell culture and repeat the cloning procedure.

Store cloned hybridomas as a seed culture (10-20 ampoules) in liquid nitrogen

tank.

6. Laboratory scale culture of the hybridoma 1. If you need small amount of monoclonal antibody for detecting antigens at

laboratory scale, culture your hybridoma in five to ten 10cm dishes to obtain

culture supernatant containing monoclonal antibody. If you need more,

spinner flask or small size hollow fiber culture apparatus would be useful for

high density culture.

2. There are many serum-free media for monoclonal antibodies’ production by

hybridomas. You can also choose protein-free media.

32

References 1. Kohler G, Milstein C., Continuous cultures of fused cells secreting antibody of

predefined specificity, Nature, 256, 495-497 (1975).

2. There are many scientific papers and books about monoclonal antibody

productions.

33

Chemical mediator release from rat basophilic leukemia RBL 2H3 cells

Introduction This is a model for Type I allergic reaction in vitro. Allergen specific IgE

antibodies bound on mast cells or basophilic cells. When allergen binds on the surface

of those cells via IgE, the cells release chemical mediators such as Histamine, Serotonin,

and proteases from granules of the cells (degranulation) to cause allergic reactions.

To investigate agricultural products having anti-allergic activity, this section

introduces a method to measure antigen mediated release of beta-hexosaminidase of

granules of rat basophilic leukemia cells with IgE class anti-DNP (dinitrophenyl)

antibody and DNP-labeled human serum albumin.

Protocol 1

Outline of the Measurement

1. Inoculate RBL 2H3 cells into a 24-well culture plate (2.5 x 10^5 cells/well) and

culture cells overnight*.

2. Incubate cells with growth medium containing 50ng/ml of mouse monoclonal

34

anti-DNP IgE antibody for 2h.

3. Wash with modified Tyrode (MT) buffer.

4. Add MT buffer containing food extract and incubate for 10 min.

5. Add DNP-labeled human serum albumin (50 ng/ml, final concentration) and

incubate for 30 min.

6. Harvest supernatants.

7. Lyse cells with MT buffer containing 0.1 % Triton X-100.

8. Determine beta-hexosaminidase activities of the supernatants and the cell lysates,

and calculate degranulation ratio.

*Basic cell culture techniques are needed.

MATERIALS

1. Medium: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with

10 % fetal calf serum (FCS)

2. Trypsin-EDTA-PBS for cell dispersion

3. PBS (Phosphate buffered saline)

4. Antibody: Anti-DNP antibody (mouse IgE class) (SIGMA D8406) Dilute

antibody with PBS to 500 microg/ml and store -30 C. Working solution: dilute

with culture medium (50 ng/ml)

5. Antigen: DNP-labeled human serum albumin (DNP-HSA, SIGMA A6661)

Dilute with PBS to 10 mg/mL and store at 4 C. Working solution: dilute with

MT buffer (1/4,000)

6. Positive control: Dissolve Wortmannin with DMSO (1 mmol/L). Dilute 1/200

with MT buffer just before use.

7. Modified Tyrode (MT) buffer: NaCl: 137 mmol/L, KCl: 2.7 mmol/L, CaCl2: 1.8

mmol/L, MgCl2: 1 mmol/L, Glucose: 5.6 mmol/L, HEPES: 20 mmol/L, BSA

(bovine serum albumin): 0.1 % Adjust pH with NaOH to 7.3. Sterilize with

filter and store at 4 C.

8. Enzyme substrate: Dissolve

p-nitrophenyl-2-acetoamido-2-deoxi-beta-d-glucopyranoside (3.3 mmol/L) with

100mmol/L citrate buffer (pH4.5)

9. Stopping solution: 2 mol/L Glycine buffer (pH 10.4, adjust pH with NaOH)

35

10. TX100 solution: Dilute Triton X-100 (0.1 %) with MT buffer.

11. Cell line: RBL-2H3 cells (rat basophilic leukemia cells) are used for the assay.

Maintain cells with 10 cm culture dish. Trypsin treatment is required to harvest

cells, because RBL-2H3 cells adhere on culture vessels strongly. However,

excess trypsinization lowers cell viability. Stop trypsin treatment before cells

floating up.

12. Samples: Dissolve samples with DMSO (up to 10%) and dilute with MT buffer

before use. Final DMSO concentration in the medium should be less than

0.1 %.

METHOD

1. Harvest RBL-2H3 cells with gentle trypsinization and centrifuge cell suspention.

Suspend cells into DMEM (2.5 x 10^5 cells/ml) and plate 1 ml of the cell

suspension into 24-well microculture plate. Culture cells at 37 C for overnight.

2. Remove medium and wash with 1 ml PBS. Add 500 micro-L anti DNP-IgE

solution and incubate for 2 h. Remove anti DNP-IgE and wash wells with 1.5 ml

MT buffer twice.

3. Add 490 micro-L MT buffer with samples or Waltomannin (control: MT buffer

only). Incubate 37 C for 10 min. Add 10 micro-L DNP-HAS and incubate at

37 C for 30 min.

4. Cool 24-well plates on ice bath and transfer supernatants from the palate to micro

sample tube. Reminded cells are lysed with 500 micro-L 0.1 % Triton X-100 by

ultrasonic treatment (cell lysates).

5. Put 50 micro-L of supernatants or cell lysates into 96-well microplate (for enzyme

assay). Add 100 micro-L of substrate solution, mix vigorously, incubate 37 C for

25 min. Stop enzyme reaction by adding 100 micro-L 2 mol/l glysin buffer.

6. Read absorbance at 405 nm and calculate beta-hexosaminidase activity.

Release Ratio (%) = 100 x [(S-Sc)/(S-Sc)+(CL-CLc)]

CL: A405nm of cell lysate

CLs: CL by adding Stopping solution first (next substrate)

S: A405nm of supernatant

Sc: S by adding Stopping solution first

36

Reference 1. Watanabe, et al., Coumarin and flavone derivatives form Estragon and Thyme as

inhibitors of chemical mediator release from RBL-2H3 cells, Biosci. Biotechnol.

Biochem., 69, 1-6 (2005)

2. Howl, et al., Intracellular delivery of bioactive peptides to RBL-2H3 cells induces

beta-hexosaminidase secretion and phospholipase D activation, Chembiochem., 4,

1312-1316 (2003).

3. Conklyn, et al., Inhibition of IgE-mediated N-acetylglucosaminidase and serotonin

release from rat basophilic leukemia cells (RBL-2H3) by tenidap: a novel

anti-inflammatory agent, Int. Arch. Allergy Appl. Immunol., 91, 369-373 (1990).

37

5. Bioassays Using Vascular Cells for Finding

Compounds that Prevent the Initiation and

Progression of Arteriosclerosis

Introduction

Vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs)

The migration and proliferation of vascular smooth muscle cells (VSMCs) and

apoptosis of vascular endothelial cells (VECs) are critically involved in the vascular

remodeling associated with the development of vascular diseases such as

arteriosclerosis and restenosis [1, 2] (see Fig. 1). Vascular endothelial injury by

various factors, such as reactive oxygen species (ROS) and increased blood pressure, is

an early event in this disease. Multiple factors including platelet-derived growth factor

(PDGF), angiotensin II, and basic fibroblast growth factor (bFGF) stimulate the

migration and proliferation of VSMCs. Therefore, molecules that can stimulate cell

growth, survival, and/or migration of VECs are candidates for preventing the

pathogenesis of arteriosclerosis by repairing the injured endothelium. Also, molecules

that can inhibit dedifferentiation, migration, and proliferation of VSMCs are candidates

to prevent the disease.

Like another several diseases, ROS play a critical role in both initiation and

progression of arteriosclerosis [3, 4]. ROS induce the apoptosis of cultured VECs

whereas they mediate the migration and proliferation of cultured VSMCs. These

findings suggest that antioxidants are strong candidates for inhibiting the development

of the disease. In fact, several natural antioxidant compounds such as resveratrol in

grapevine and epigallocatechin gallate in green tea that can scavenge ROS are known to

prevent the development of arteriosclerosis.

In the following protocols, medium containing high concentrations of glucose

(4500 mg/ml) and medium containing low concentrations of glucose (1000 mg/ml) are

used, depending on the purpose of each experiment. That is, high-glucose medium

38

reflects the state of hyperglycemia whereas low-glucose medium indicates the normal

state. In fact, the concentration of glucose affects various intracellular signaling

pathways and the expression of various genes in cultured VECs and VSMCs. Diabetes

is a well-known risk factor for the pathogenesis of arteriosclerosis. Therefore, we

should select the appropriate medium depending on the purpose of each experiment.

Protocol 1 Wound healing assay of VECs.

The wound healing of VECs consists of two cellular responses, proliferation and

migration of these cells. Thus, if we investigate the effect of some compound on the

wound healing in a short time upon stimulation (e. g. 8 hours), the result is dependent

on its migration-stimulating activity. On the other hand, if we examine the effect in a

long time after stimulation (e. g. 1 day), the result reflects its proliferation- and/or

migration-stimulating activity. We recommend a long-time wound-healing analysis as

a first step for screening useful compounds. This assay system can also be used to find

compounds that prevent the metastasis of cancer as well as the development of

arteriosclerosis.

MATERIALS

Cells: Porcine VECs*1

Medium and Solutions:Dulbecco’s modified Eagle’s medium (DMEM, high

glucose 4500 mg/l) supplemented with 10% fetal calf serum (FCS), 100 units/ml

penicillin and 100 µg/ml streptomycin

DMEM (low glucose 1000mg/l) supplemented with 10% fetal calf serum

(FCS), 100 units/ml penicillin and 100 µg/ml streptomycin

DMEM (high glucose) supplemented with 1% FCS, 100 units/ml penicillin and 100

g/ml streptomycin

DMEM (low glucose) supplemented with 1% FCS, 100 units/ml penicillin and

100 g/ml streptomycin

Phosphate-buffered saline without Ca2+ (PBS(-))

FCS *2

39

*1: Porcine VECs are prepared from the thoracic aorta (see Protocol 6) and used

between passages 5 and 10.

*2: The property of porcine VECs is sometimes affected by FCS. We need to

check several lots of FCS for finding FCS suitable for the culture of porcine

VECs.

METHOD

1. Inoculate cells in a 3.5-cm dish (2.0 x 105 cells/ml/dish).

2. Culture cells in DMEM supplemented with 10% FCS and the antibiotics for

6-8 h

3. Wash cells with PBS(-) once.

4. Add 1 ml of DMEM supplemented with 1% FCS and the antibiotics.

5. Culture cells for 16 h.

6. Add each sample*3, *4 and culture cells for 30 min.

7. Create a wound using a 200-l tip (see Fig. 2).

8. Culture cells for 8 h and 24 h for a short-time and a long-time analysis,

respectively.

9. Take a photograph (see Fig. 2).

*3: The volume of each sample added should be up to 1/100 of that of medium to

avoid the influence of vehcle on VECs.

*4: Basic fibroblast growth factor (bFGF) can be used as a positive control for both

proliferation and migration. On the other hand, lysophosphatidic acid (LPA) is

convenient as a positive control for migration because LPA can induce

migration, but not proliferation of VECs.

Protocol 2 Cell proliferation assay of VECs.

Various kinds of proliferation assay kits are commercially available in recent years.

Most of the kids evaluate the cell number by measuring the activity of some

mitochondrial enzymes. This protocol also uses this kind of kit. We basically follow

a manufacture’s instruction of the kit. This assay protocol can be used for most of

40

another cells including VSMCs as well as VECs. As Protocol 1, this protocol can also

be applied for finding compounds that inhibit the metastasis of cancer.

MATERIALS

Cells, medium, and solutions are the same as those in Protocol 1.

Proliferation assay kit: CellTiter 96AQueous One Solution Cell Proliferation Assay

(Promega)*1

METHOD

1. Inoculate cells in a 96-well dish (3.0 x 103 cells/well).

2. Culture cells in DMEM supplemented with 10% FCS and the antibiotics for

6-8 h.

3. Wash cells with PBS (-) once.

4. Add 100 l of DMEM supplemented with 1% FCS and the antibiotics


6. Add each sample*2 and culture cells for 30 min.

7. Culture cells for another 24 h.

8. Wash cells with PBS(-) twice.


10. Add 20 µl of CellTiter 96AQueous One Solution.

11. Incubate the mixture at 37 for 1.5 h.

12. Measure the absorbance at 492 nm.

*1: The CellTiter 96AQueousOne Solution Cell Proliferation Assay is a

colorimetric

Method for determining the number of viable cells in proliferation or

cytotoxicity assays. The CellTiter 96AQueousOne Solution Reagent contains a

novel tetrazolium compound

[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2

H- tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine

ethosulfate; PES).

*2: Basic fibroblast growth factor (bFGF) can be used as a positive control for

41

proliferation of VECs.

Protocol 3 Cell migration assay of VSMCs.

PDGF is critically involved in the pathogenesis of arteriosclerosis by inducing

proliferation and migration of VSMCs. We use this protocol to find compounds that

inhibit PDGF-induced migration of VSMCs. If we add angiotensin II, which is also

crucially implicated in the pathogenesis of hypertension and arteriosclerosis, instead of

PDGF, we can look for compounds that inhibit angiotensin II-stimulated VSMCs

migration.

Cell migration in this protocol is determined using a modified Boyden chamber

assay. The principle of this assay is as follows. At first polyvinylpyrrolidone-free

polycarbonate membranes with a pore size of 8 m are coated with fibronectin.

Serum-starved VSMCs are pre-incubated with or without various compounds of interest,

and added to the upper chamber (transwell). The upper chambers with cells are

inserted into the bottom chamber filled with medium, and incubated in the presence and

absence of stimulators such as PDGF and angiotensin II for 5 h. Cells that migrate to

the bottom surface of the membrane are fixed by methanol, stained with hematoxylin,

and counted in five to ten representative fields.

MATERIALS

Cells: rat VSMCs*1 (see Protocol 7)

Polyvinylpyrrolidone-free polycarbonate chamber membrane: Transwell (Corning,

Cat. No. 3422, 6.5-mm diameter membrane, 8-m pore size)

Medium and Solutions:

DMEM (high glucose 4500 mg/l) supplemented with 10% FCS, 100 units/ml

penicillin and 100 g/ml streptomycin

DMEM (low glucose 1000 mg/l) supplemented with 10% FCS, 100 units/ml


DMEM (high glucose 4500 mg/l) supplemented with 0.1% FCS, 100 units/ml


DMEM (low glucose 1000 mg/l) supplemented with 0.1% FCS, 100 units/ml

42


PBS(-)

TE

*1: Rat VSMCs are prepared from the thoracic aorta (see Protocol 7) and used

between passages 5 and 10.

METHOD

1. Culture cells in DMEM supplemented with 0.1% FCS and the antibiotics for 48

h in a 10-cm dish.

2. Coat a 6.5-mm polycarbonate membrane in a transwell with fibronectin (10

µg/ml) for at least 30 min.

3. Culture cells with each sample*2 for 30 min during the step 2.

4. Inoculate pre-treated cells on the fibronectin-coated membrane in the transwell

(upper chamber) (2 x 105 cells/chamber) and insert it into a 24-well dish

(bottom chamber) filled with DMEM supplemented with 0.1% FCS and the

antibiotics.

5. Add PDGF (20 ng/ml) to the bottom chamber and add each sample at the same

concentration of step 3 in the both chamber.


7. Remove the medium, add methanol, and leave the membrane for 2 min at room

temperature.

8. Remove methanol, add hematoxylin, and leave the membrane for 1-2 days.

9. Remove the membrane from the transwell and count migrated cells on the

bottom surface under a microscope.

*2: We use the PDGF receptor inhibitor AG14785 as a positive control for inhibiting

PDGF-induced migration.

Protocol 4 Cell proliferation assay of VSMCs.

This protocol uses the same kit in Protocol 2. Thus, most of experimental steps

43

are the same as those in Protocol 2. However, whereas Protocol 2 is for finding

compounds that stimulate the proliferation of VECs, this protocol is to find compounds

that inhibit the proliferation of VSMCs.

MATERIALS

Cells: rat VSMCs (see Protocol 7)


DMEM (high glucose 4500 mg/l) supplemented with 10% FCS, 100 units/ml


DMEM (low glucose 1000 mg/l) supplemented with 10% FCS, 100 units/ml


DMEM (high glucose 4500 mg/l) supplemented with 0.1% FCS, 100 units/ml


DMEM (low glucose 1000 mg/l) supplemented with 0.1% FCS, 100 units/ml


PBS(-)

TE

Proliferation assay kit: CellTiter 96AQueous One Solution Cell Proliferation Assay

(Promega)*1

*1: See Protocol 2.

METHOD

1. Inoculate cells in a 96-well dish (2.0 x 103 cells/ well).

2. Culture cells in DMEM (high glucose) supplemented with 10% FCS and

antibiotics for 3 days.

3. Wash cells with PBS (-) once.

4. Culture cells in DMEM supplemented with 0.1% FCS and antibiotics for 2 days.

5. Add each sample*2 and culture cells for 30 min.

6. Add PDGF (20 ng/ml).

7. Culture cells for 2 days.

8. Wash cells with PBS (-) twice.


44

10. Add 20 l of CellTiter 96AQueous One Solution.

11. Incubate the mixture at 37 for 1 h.

12. Measure the absorbance at 492 nm.

*2: We use the PDGF receptor inhibitor AG14785 as a positive control for inhibiting

PDGF-induced cell proliferation,

Protocol 5

Maintenance of VECs and VSMCs MATERIALS

Cells: Porcine VECs (see Protocol 6) and rat VSMCs (see Protocol 7).


DMEM (high glucose 4500 mg/l) supplemented with 10% FCS*1 and antibiotics

PBS (-)

0.05% Trypsin/0.02% EDTA (TE)

*1: The property of porcine VECs is sometimes affected by FCS. We need to

check several lots of FCS for finding FCS suitable for the culture of porcine

VECs.

METHOD

1. Culture cells in DMEM supplemented with 10% FCS and antibiotics. We

usually maintain VECs and VSMCs in a 10-cm dish.

2. Change culture medium every 2-3 days.

3. Keep culturing until cells become confluent.

4. Wash cells with PBS (-) twice.

5. Add 1 ml of TE and incubate for 2-5 min to detach cells.

6. Add 10 ml of DMEM supplemented with 10% FCS and antibiotics to stop the

trypsin reaction.

7. Collect cells by centrifugation at ~1000 x g.

8. Suspend cells in an appropriate volume of DMEM supplemented with 10% FCS

and antibiotics.

45

9. Count cell number using trypan blue.

10. Inoculate cells 1:4 into a new 10-cm dish for cell maintenance.

Protocol 6

Preparation of porcine VECs VECs are prepared from the thoracic aorta of prepubertal pigs.

MATERIALS

Medium 1: DMEM (high glucose 4500 mg/) supplemented with 10% FCS, 100

units/ml penicillin, and 100 µg/ml streptomycin

Medium 2: DMEM (high glucose 4500 mg/) supplemented with 100 units/ml


PBS(-)

Solution 1: PBS (-) containing 300 units/ml penicillin and 300 µg/ml streptomycin

METHOD*1

1. Collect several fragments of the thoracic aorta (10-15 cm long) of prepubertal

pigs slaughtered at a local abattoir.

2. Wash them with 500 ml of PBS (-) and soak them in Solution 1.

3. Remove connective tissue and lipid around the aorta.

4. Cut the aorta with scissors along the long axis to open the aorta.

5. Fix the opened aorta inside out (the intima is upper side) on Styrofoam covered

with aluminum foil.

6. Remove the surface layer (intima) of the aorta with scalpel and soak the scalpel

with the intima into 15-ml centrifugation tube filled with 5 ml of Medium 2 (one

fragment of aorta/tube). The intima layeres are dispersed.

7. Tap the tube well to loosen cells.

8. Centrifuge the tube at ~1000 x g for 5 min.

9. Remove the supernatant and add 5 ml of Solution 1 to cells.

10. Disperse cells completely with pipette.

11. Centrifuge the tube at ~1000 x g for 5 min.

12. Collect and inoculate cells in a 10-cm dish filled with Medium 1.

46

*1: Every step is performed at room temperature.

Protocol 7

Preparation of porcine VSMCs VSMCs are prepared from the thoracic aorta of adult male Sprague-Dawley rats.

MATERIALS

Animal: Adult male Sprague-Dawley rat

Reagents:

Albumin from bovine serum (BSA)

Collagenase type 1

Trypsin inhibitor

Elastase type III

HEPES

Medium 1: DMEM (high glucose, 4500 mg/l)

Medium 2: DMEM (high glucose, 4500 mg/l) containing 300 units/ml penicillin

300 µg/ml streptomycin

Medium 3: DMEM (high glucose, 4500 mg/l) supplemented with 10% FCS, 100

units/ml penicillin and 100 µg/ml streptomycin

Solution 1: PBS (-) containing 300 units/ml penicillin and 300 µg/ml

streptomycin

Solution 2: Solution 2 (10 ml) contains 100 mg HEPES, 20 mg BSA, 10 mg

collagenase type 1, 3.75 mg trypsin inhibitor, and 1.25 mg elastase in

Medium 1. This solution is also freshly prepared for each

experiment.

Solution 3: Solution 3 (1 ml) contains 1 mg collagenase type 1 in Medium 2.

METHOD (for one rat)*1

1. Remove the thoracic aorta from an anesthetized rat with ether and soak it in a

6-cm dish filled with ~10 ml of Solution 1.

2. Remove connective tissue and lipid around the aorta.

47

3. Transfer the aorta into a 15-ml centrifugation tube filled with 3 ml of Solution 2.

4. Shake it at 37 for 30 min at a speed of 120-160 rpm*2.

5. Transfer the aorta into a 6-cm dish filled with 3 ml of Solution 1 and remove the

adventitia of the aorta.

6. Transfer the aorta into a 3.5-cm dish and add 1 ml of Solution 3.

7. Cut the aorta with scissors into small pieces.

8. Transfer all of the pieces into a 15-ml centrifugation tube using another 4-5 ml

of Solution 3.

9. Shake the tube at 37 for over 120 min at a speed of 160 rpm.

10. Add 4 ml of Medium 3 and centrifuge it at ~1000 x g for 5 min.

11. Remove the supernatant and inoculate cells in 3.5-cm collagen type 1-coated

dish*3.

*1: Every step is performed at room temperature.

*2: Do not extend the incubation period over 30 min.

*3: VSMCs, but not VECs bind collagen-coated dish and spread. A 6-cm and

10-cm dishes are used for the second and third passage, respectively.

Collage-coated dishes are essential for the first and second passage. If the

preparation goes well, we can see adherent cells next day.

References

1. Newby, A. C., Zaltsman, A. B. (2000) Molecular mechanisms in intimal

hyperplasia. J. Pathol. 190, 300-309.

2. Heldin, C. H., and Westermark, B. (1999) Mechanism of action and in vivo role of

platelet-derived growth factor. Physiol. Rev. 79, 1283-1316.

3. Cai H, Griendling KK, Harrison DG. (2003) The vascular NAD(P)H oxidases as

therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci. 24, 71-478.

4. Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. (2003) Role of

oxidative stress in atherosclerosis. Am J Cardiol. 91, 7A-11A.

Figure 1. Model of the pathogenesis of arteriosclerosis

48

Stimulator + Stimulator –

Figure 2. Model of the pathogenesis of arteriosclerosis

Figure 3. Transwell

49

6. Culture of Bone-Related Cells and Analysis of

Calcification

Introduction Bone is a dynamic tissue that undergoes continuous remodeling. In the mature

adult, lost bone is replaced by an equivalent amount of formation of newly formed bone,

with resultant preservation of the integrity of the skeleton. Bone remodeling involves

the complex and tightly coordinated actions of bone-resorbing osteoclasts and

bone-forming osteoblasts (Figure 1). When the balance of activities between

osteoblasts and osteoclasts were destroyed, we caused osteoporosis.

Figure 1. Bone remodeling

Osteoblasts are bone-forming cells. The formation of bone involves a complex

series of events that include the proliferation and differentiation of osteoprogenitor cells

and result eventually in the formation of a mineralized extracellular matrix. Several

model systems have been developed for studies of the proliferation and differentiation

of bone-forming cells in vitro and the molecular biology of the mineralization process

(1-5). The sequential expression of type I collagen, ALPase (alkaline phosphatase) and

50

osteocalcin, and the deposition of calcium are known as markers of osteoblastic

differentiation.

Osteoclasts are multinuclear cells that are responsible for resorption of bone.

The osteoclastic resorption of bone entails several processes: the development of

osteoclasts from hematopoietic progenitor cells; the fusion of osteoclasts; and the

secretion of acids and lysosomal enzymes into the space beneath osteoclasts [6]. The

formation and activation of osteoclasts are controlled by the combined action of

RANKL (receptor activator of nuclear factor-κB ligand) [7-10], OPG (osteoprotegerin)

[11,12], and M-CSF (Macrophage-colony stimulating factor) [7] produced by

osteoblasts or osteogenic stromal cells (e.g. ST2 cells). 1 α, 25 (OH)2 vitamin D3

stimulates and inhibits the expression of mRNAs for RANKL and OPG, respectively

[7-12]. We used here cocultures with mouse spleen cells (osteoclast precursor cells)

and ST2 cells (osteoclast-supporting cells) in a well-characterized model system that

has been used for studies of osteoclast differentiation, fusion, and resorption activity

[13-15].

Protocol 1

Osteoblast Culture MATERIALS

Cells

MC3T3-E1 cells; mouse calvarial preosteoblastic cells (RIKEN CELL BANK,

Tsukuba, Japan)

Buffers and Solutions

α-MEM; Minimum Essential Medium Eagle Alpha Modification

(Sigma-Aldorich)

FBS; Fetal bovine serum (Moregate, Australia & New Zealand)

Penicillin-Streptomycin Solution; 50 units/ml penicillin and 50 µg/ml

streptomycin

(GIBCO Invitrogen Corp.)

0.05% Trypsin solution

Trypsin (Type V-S from Bovine Pancreas, SIGMA) is dissolved with PBS; 20 mM

sodium phosphate and 130 mM NaCl, pH 7.4, and filtered with 0.22 µm, stored at

51

–20 C until use.

Special Equipment

Cleanbench

CO2-incubator

100 mm Tissue Culture Dish and Microplate (12 Well/Flat Bottom)

METHOD

All procedures are done in Cleanbench and hand and ware are sterilized with 70%

ethanol.

1. Cells are maintained in 55-cm2 dishes in α-MEM, supplemented with 10% fetal

bovine serum, 50 units/ml penicillin and 50 µg/ml streptomycin, in a humidified

atmosphere of 5% CO2 in air at 37 C.

2. After cells had reached 70% confluence, cells are detached by treatment with

0.05% trypsin, replated in either 100 mm culture dishes or 12-well plates (area of

each well, 3.8 cm2) at a density of 1 × 104 cells/cm2 and grown in calcified

medium (α-MEM supplemented with 10% fetal bovine serum, 50 units/ml

penicillin, 50 µg/ml streptomycin, 5 mM β-glycerophosphate, 50 mg/ml ascorbic

acid).

3. Fresh medium was supplied to cells at 3-day intervals. Samples were dissolved

in ethanol or DMSO; dimethylsulfoxide (final concentration of 0.1%) and 0.1%

ethanol and DMSO do not affect the proliferation and differentiation of

MC3T3-E1 cells.

Protocol 2

Quantitation of the Deposition of Calcium MATERIALS


Phosphate-buffered saline

pH 7.4; PBS; 20 mM sodium phosphate and 130 mM NaCl

2N HCl

52

Special Equipment

Spectrophotometer

Shaker

Additional Regents

Calcium C Test Wako kit (Wako Pure Chemical Industries, Osaka, Japan).

METHOD

1. Prepare the cultures for measurement of deposition of calcium:

Osteoblastic cells (MC3T3-E1 cells) were subcultured in 12-well plates (3.8

cm2/well) in α-MEM that contained 10% fetal bovine serum, 5 mM

ß-glycerophosphate, 50 µg/ml ascorbic acid, and samples at various concentrations.

Calcification was observed at the culture approximately for 20 days.

2. The amount of calcium, in hydroxyapatite, in cell layers was measured as follows.

The layers of cells in 12-well plates were washed twice with 1 ml PBS and

incubated with 1 ml of 2 N HCl overnight with gentle shaking.

3. The Ca2+ ions in samples were quantitated by the o-cresolphthalein complexone

method with a Calcium C Test Wako kit (Wako Pure Chemical Industries, Osaka,

Japan).

Protocol 3

Osteoclast Culture and Tartrate-Resistant Acid Phosphatase (TRAP) Staining MATERIALS

Animals

ddY male mouse from 6 to 9 week


Phosphate-buffered saline (filtered with 0.22 µm filter unit)

pH 7.4; PBS; 20 mM sodium phosphate and 130 mM NaCl (store at 4 C)

Hemolytic solution (filtered with 0.22 µm filter unit)

0.83% NH4Cl : 10 mM Tris-HCl (Ph 7.4) = 9 : 1 (store at 4 C)

α-MEM; Minimum Essential Medium Eagle Alpha Modification

53

(Sigma-Aldorich)

10% Formalin (final 3.7% Formaldehyde solution)

Ethanol : Aceton = (1 : 1)

TRAP solution (store at 4 C and use within one week)

Naphtol AS-MX phosphate 5 mg and Fast red LB solt 30 mg were dissolved in 0.5

ml N-N-DMF, and then added 0.1 M sodium acetate (pH 5.0) containing 50 mM

sodium tartrate.

Special Equipment

Cleanbench

CO2-Incubator

Syringe (1 ml)

Cell Strainer; 70 µm (BD Falcon; Bedford, MA, USA)

Syringe Driven Filter Unit; PVDF 0.22 µm (MILLEX-GU, MilliPORE; Cork,

Ireland)

Centrifuge

Dryer

Additional Regents

sRANKL (soluble) osteoclast-differentiating factor

M-CSF (Macrophage-colony stimulating factor) osteoclast-differentiating

factor

METHOD

Osteoclast Culture; All procedures are done in cleanbench and hand and ware are

sterilized with 70% ethanol.

1. Prepare an anesthetized mouse (six-week old male ddY mouse) and rinse with

70% ethanol.

2. Spleen is removed from mouse and washed with PBS. Splenic tissues are

pounded up with the bottom of 1 ml syringe and pipetting. Spleen cell solution

is filtered with Cell Strainer.

3. The solution is centrifuged at 1,000 rpm for 5 min and pellet is resuspended

with 1 ml Hemolytic solution to eliminate erythrocytes contaminating the spleen

cell fraction. The solution is centrifuged at 1,000 rpm for 5 min and

54

resuspended with 5 ml PBS. The solution is centrifuged at 1,000 rpm for 5

min and pellet is resuspended with 10 ml α-MEM containing 10% fetal bovine

serum.

4. Spleen cells are counted under a microscope. (approximately 108 cells/a mouse)

5. Prepare the microplate (96-well plate).

6. 150 µl Spleen cells (2.4 × 105 cells/well) in 96-well plates (0.32 cm2/well) are

cultured with 50 ng/ml human sRANKL and 30 ng/ml M-CSF for 6 to 8 days.

7. The cultures are maintained at 37 C in a humidified atmosphere of 5% CO2 in

air. Fresh medium and samples are supplied at 2-day intervals.

TRAP Staining

1. Prepare osteoclasts subcultured for 6 to 8 days.

2. Adherent cells are fixed in 3.6% formaldehyde for 5 min and then in a mixture

of ethanol and acetone (1:1, v/v) for 1 min. Cells are dried with a dryer.

3. Fixed osteoclasts are stained for TRAP activity (Figure 2). TRAP solution

(100 µl/well; 96-well plate) is added to the culture and the plate is shaked at 37

C for 10 to 15 min. TRAP solution is removed. This process repeats once

more. Then the wells are washed twice with H2O.

4. TRAP-positive mononuclear cells and TRAP-positive multinucleated cells

(with three or more nuclei) are counted under a microscope (IX70; Olympus,

Tokyo, Japan).

Figure 2. TRAP-positive multinucleated osteoclastic cells

55

References 1. Hagiwara H, Inoue A, Yamaguchi A, Yokose S, Furuya M, Tanaka S, Hirose S 1996

cGMP produced in response to ANP and CNP regulates proliferation and

differentiation of osteoblastic cells. Am J Physiol 270:C1311-C1318

2. Inoue A, Hiruma Y, Hirose S, Yamaguchi A, Furuya M, Tanaka S, Hagiwara H 1996、

Stimulation by C-type natriuretic peptide of the differentiation of clonal osteoblastic

MC3T3-E1 cells. Biochem Biophy Res Commun 221:703-707

3. Bresford JN, Graves SE, Smoothy CA 1993 Formation of mineralized nodules by

bone derived cells in vitro: a model of bone formation? Am J Med Genet

45:163-178

4. Liu F, Malaval L, Gupta AK, Aubin JE 1994 Simultaneous detection of multiple

bone- related mRNAs and protein expression during osteoblast differentiation:

polymerase chain reaction and immunocytochemical studies at the single cell level.

Dev Biol 166:220-234

5. Stein GS, Lian JB, Owen TA 1990 Relationship of cell growth to the regulation of

tissue-specific gene expression during osteoblast differentiation. FASEB J

4:3111-3123

6. Suda T, Nakamura I, Jimi E, Takahashi N 1997 Regulation of osteoclast function. J

Bone Miner Res 12:869-879

7. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux

ER, Teepe MC, DuBose RF, Cosman D, Galibert L 1997 A homologue of the TNF

receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature

390:175-179

8. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E,

Bartlett FSI, Frankel WN, Lee SY, Choi Y 1997 TRANCE is a novel ligand of the

tumour necrosis factor receptor family that activates c-Jun N-terminal kinase in T

cells. J Biol Chem 272:25190-25194

9. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S,

Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K,

Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand

for osteoprotegerin/osteoclastogenesis-inhibitory factor and identical to

TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597-3602

10. Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R,

56

Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli

C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J,

Boyle WJ 1998 Osteoprotegerin ligand is a cytokine that regulates osteoclast

differentiation and activation. Cell 93:165-176

11. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ,

Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A,

Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshow-Gegg L, Hughes TM, Hill

D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Amgen EST

Program, Boyle WJ 1997 Osteoprotegerin: a novel secreted protein involved in the

regulation of bone density. Cell 89:309-319

12. Yasuda H, Shima N, Nakagawa N, Mochizuki S, Yano K, Fujise N, Sato Y, Goto M,

Yamaguchi K, Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio

K 1998 Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin

(OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro.

Endocrinology 139:1329-1337

13. Udagawa N, Takahashi N, Akatsu T, Sasaki T, Yamaguchi A, Kodama H, Martin TJ,

Suda T 1989 The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2

support osteoclast-like cell differentiation in cocultures with mouse spleen cells.


14. Otsuka E, Kato Y, Hirose S, Hagiwara H 2000 Role of ascorbic acid in the

osteoclast formation: induction of osteoclast differentiation factor with formation of

extracellular collagen matrix. Endocrinology 141:3006-3011

15. Yamagishi T, Otsuka E, Hagiwara H 2001 Reciprocal control of expression of

mRNAs for osteoclast differentiation factor and OPG in osteogenic stromal cells by

genistein: evidence for the involvement of topoisomerase II in osteoclastogenesis.


57

7. Working with Adipocytes

Adipocyte as an endocrine cell

Adipose tissue has been ignored by anatomists and physicians for centuries,

considered to be just an energy storage depot. The well-documented rise in obesity

during the past 30 years [1] has contributed to the negative image of adipose tissue.

The past two decades, however, have seen a wave of intense scientific interest in

adipocytes, fuelled in part by concerns about obesity and its attendant metabolic

diseases [2], and also by the recognition that adipocytes integrate a wide array of

homeostatic processes. In addition to regulating fat mass and nutrient homeostasis,

adipocytes are involved in the immune response, blood pressure control, haemostasis,

bone mass, and thyroid and reproductive function [3]. These processes are coordinated

mainly through the synthesis and release of peptide hormones, so-called adipocytokines

or adipokines, by adipocytes [4-5].

Although most multicellular organisms have cells that store excess energy,

adipocytes evolved to meet this need at the time of the vertebrate radiation. Mammals,

birds, reptiles, amphibians and many fish have cells that are readily identifiable as

adipocytes, although the anatomical location of fat tissues varies considerably between

species. Most mammals have stereotypical adipose depots located throughout the

body. Some of these depots are thought to be largely structural, providing mechanical

support but contributing relatively little to energy homeostasis. There are several

distinct depots within the body cavity, surrounding the heart and other organs,

associated with the intestinal mesentery, and in the retroperitoneum. Some of these

depots, known as visceral fat, drain directly into the portal circulation and have been

linked to many of the morbidities associated with obesity, including type 2 diabetes and

cardiovascular disease. Adipocytes and precursor cells from different depots have

different replicative potential, different developmental attributes and different responses

to hormonal signals, although the mechanistic basis for these distinctions is still unclear

[6].

58

Transcriptional regulation of adipocyte differentiation

Adipocytes have been a popular model for the study of cell differentiation, since

the development of the murine adipose 3T3 cell culture system by Green and colleagues

[7]. There have been several thorough reviews on this aspect of adipose biology

recently [8-9], so we present only the core of this regulatory system. The central

engine of adipose differentiation is peroxisome proliferator-activated receptor-g

(PPAR-g) [10-12]. When this receptor is activated by an agonist ligand in fibroblastic

cells, a full program of differentiation is stimulated, including morphological changes,

lipid accumulation and the expression of almost all genes characteristic of fat cells.

Multiple CCAAT/enhancer-binding proteins (C/EBPs) also have a critical role in

adipogenesis, with C/EBP-b and C/EBP-d driving PPAR-g expression in the early

stages of differentiation and C/EBPa maintaining PPAR-g expression later on in the

process [8]. C/EBPs and PPAR-g also directly activate many of the genes of

terminally differentiated adipocytes. More recently, other factors have been implicated

in the differentiation process, including several Krüppel-like factors [13-15], early

growth response 2 (Krox20) [16] and early B-cell factors [17].

ROS production in adipocytes

Oxidative stress plays critical roles in the pathogenesis of various diseases [18].

In the diabetic condition, oxidative stress impairs glucose uptake in muscle and fat

[19-20] and decreases insulin secretion from pancreatic b cells [21]. Increased oxidative

stress also underlies the pathophysiology of hypertension [22] and atherosclerosis [23]

by directly affecting vascular wall cells. It has recently been suggested that obesity per

se may induce systemic oxidative stress and that increased oxidative stress in

accumulated fat is, at least in part, the underlying cause of dysregulation of

adipocytokines and development of metabolic syndrome [24]. As an early instigator

of obesity-associated metabolic syndrome, increased oxidative stress in accumulated fat

should be an important target for the development of new therapies.

59

Protocol 1

Cell culture and adipogenic differentiation of 3T3-L1 cells

This protocol was originally reported by Green and colleagues7 and has been used

as a conventional method. 3T3-L1 is a cell line derived from Swiss 3T3 cells and most

frequently used for in vitro adipogenic differentiation. Using this cell culture system,

samples of interest can be assayed for inhibiting or enhancing adipogenic differentiation

process.

Preadipocyte Adipocyte (Day10)

MATERIALS

Cells

3T3-L1 cells (ATCC number: CCL92.1)


Dulbecco’s modified Eagle’s medium (DMEM, high glucose 4500mg/L);

commercially available from Sigma or other companies.

Phosphate buffered saline (PBS)

0.05% Trypsin/0.02% EDTA

Insulin (Sigma I-1882);

dissolve in sterile (0.2µm filtered) 1% acetic acid solution, and store at -20°C and 4°C

for stock and working solution, respectively.

Dexamethasone (Dex. Sigma D-4902);

dissolve in 100% ethanol (solubility 1mg/mL in ethanol), and store at -20°C

methyl-3-isobutylxanthine (MIX, Sigma I-5879)

dissolve in 100% ethanol (solubility 10mg/mL in ethanol), and store at -20°C

featal calf serum (FCS)

60

METHOD

1. Culture 3T3-L1 cells in DMEM supplemented with 10% FCS*1.

2. Change culture medium every 2-3 days

3. Keep culturing until cells become confluent

4. Change culture medium and culture for another 2 days for clonal expansion *2.

5. Change medium with differentiation induction one (culture medium supplemented

containing 0.25µM Dex, 0.5 mM MIX, 10 µg/mL insulin) with or without sample

of interest for 48 h.

6. Change culture medium with 5 µg/mL insulin with or without sample of interest

every 2 days and keep culturing until lipid droplets become visible (usually 8 to 10

days after differentiation induction).

*1What is most important with this protocol is that cells must not be allowed to

become confluent during passage culture. When cell density becomes around 60

to 70%, immediately detach cells by trypsinization and inoculate cells 1:5 into

new cell culture dishes. It is highly recommended to have cell stocks before

individual experiments. If you have less differentiated cells, restart with a new

stock of cells.

*2This medium change is essential for maximal differentiation.

Protocol 2

Oil red O staining of 3T3-L1 adipocytes Oil red O stain lipid droplets stored in differentiated adipocytes. This method is

an easiest one to evaluate a degree of adipogenic differentiation in culture.

MATERIALS

Cells



PBS

61

Formalin

2-propanol

Oil red O solution;

Dissolve 150mg Oil red O (Sigma O-0625) in 50 mL 2-propanol with

continuous shaking for 10 min. This solution must be used within 2 h after

preparation.

METHOD

1. Wash cells with PBS twice

2. Fix cells with 10% formalin at room temperature for 10 min and wash with PBS

twice.

3. Add 60% (v/v) 2-propanol and incubate at room temperature for 1 min.

4. Add Oil red O solution and incubate at room temperature for 20 min.

5. Wash cells with 2-propanol once and with PBS twice.

6. Observe cells by microscopy and take photographs if necessary*1.

7. Stained Oil red O can be extracted with 90% (v/v) of 2-propanol containing 1%

NP-40 (0.5 mL for a well of 12-multiwell plate). Determine absorbance at 490

nm to evaluate quantitatively a degree of adipogenic differentiation.

*1Typical staining pattern is shown below. Pictures were taken 0, 2, and 8 days

after differentiation induction (from left to right).

Protocol 3

Determination of ROS production in 3T3-L1 adipocytes

Original protocol was reported by Oliveira et al. using pancreatic b-cells25 and

applied for adipocytes24. Using this protocol, samples with potential anti-oxidative

activity can be assayed for adipocytes as well as other types of cells.

62

MATERIALS

Cells



PBS

Nitroblue tetrazolium (NBT, Sigma N-6639)

METHOD

1. Induce adipogenic differentiation of 3T3-L1 cells in a 12-well plate, as described

above.

2. Wash cells with PBS twice

3. Incubate cells for 90 min in 0.5 mL of PBS containing 0.2% NBT*1 in a CO2

incubator with or without sample of interest.

4. Observe insoluble formazan formed during incubation*2.

5. Dissolve the formazan in 0.5 mL of 50% acetic acid

6. Determine absorbance at 560 nm*3.

*1 PBS containing 0.2% NBT should be freshly prepared. If you find some

precipitates in the solution, spin down briefly and use supernatant for an assay. *2 It is possible to stop incubation before 90 min if formazan formation is enough. *3 It is convenient to use 96-multiwell plate if multiple samples are assayed.

Reference

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the metabolic syndrome. Endocrine 29, 109-17 (2006).

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fat. Acta Physiol Scand 184, 285-93 (2005).

4. Matsuzawa, Y. White adipose tissue and cardiovascular disease. Best Pract Res Clin

Endocrinol Metab 19, 637-47 (2005).

5. Koerner, A., Kratzsch, J. & Kiess, W. Adipocytokines: leptin--the classical,

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resistin--the controversical, adiponectin--the promising, and more to come. Best

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7. Green, H. & Kehinde, O. An established preadipose cell line and its differentiation

in culture. II. Factors affecting the adipose conversion. Cell 5, 19-27 (1975).

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(2006).

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adipocyte differentiation. Cell Metab 1, 27-39 (2005).

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of adipogenesis. J Biol Chem 280, 12867-75 (2005).

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19. Maddux, B. A. et al. Protection against oxidative stress-induced insulin resistance

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20. Rudich, A. et al. Prolonged oxidative stress impairs insulin-induced GLUT4

translocation in 3T3-L1 adipocytes. Diabetes 47, 1562-9 (1998).

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