Page 1 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM BMS 631 - LECTURE 13 Flow Cytometry:...

©1990-2013 J.Paul Robinson, Purdue University09:36 PM

BMS 631 - LECTURE 13 Flow Cytometry: Theory

Bindley Bioscience CenterOffice: 765-494 0757email; [email protected]

Cell Function

J. Paul RobinsonSVM Professor of Cytomics &Professor of Biomedical EngineeringPurdue University

WEB http://www.cyto.purdue.edu

Notice: The materials in this presentation are copyrighted materials. If you want to use any of these slides, you may do so if you credit each slide with the author’s name. It is illegal to copy these to CourseHero or any other online theft system.


Cellular Response

• Cell death• Cell ‘suicide’• Ignore damage• Damage repair• Incorrect repair


Functional Assays

•intracellular pH•intracellular calcium•intracellular glutathione•oxidative burst•phagocytosis


Oxidative Burst

•generation of toxic oxygen speciesby phagocytic cells

•superoxide anion measuredwith hydroethidine

•hydrogen peroxide measured with2’,7’-dichlorofluorescin diacetate(DCFH-DA)


TIME (seconds)

2400 1800 1200 600

Scale

34511538

124

Neutrophil Oxidative Burst

PMA-StimulatedNeutrophils

UnstimulatedNeutrophils

0

Log

DC

F1

1010

010

00


FITC-Labeled Bacteria

Phagocytosis


Cellular Functions

• Cell Viability• Phagocytosis• Organelle Function

– mitochondria, ER– endosomes, Golgi

• Oxidative Reactions– Superoxide– Hydrogen Peroxide– Nitric Oxide– Glutathione levels

• Ionic Flux Determinations–Calcium

–Intracellular pH

• Membrane Potential

• Membrane Polarization

• Lipid Peroxidation


Organelle Function

• Mitochondria Rhodamine 123• Endosomes Ceramides• Golgi BODIPY-Ceramide

• Endoplasmic Reticulum DiOC6(3)

Carbocyanine


Fluorescent Indicators

How the assays work:• Superoxide: Utilizes hydroethidine the sodium borohydride

reduced derivative of EB• Hydrogen Peroxide: DCFH-DA is freely permeable and enters

the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent DCF

• Glutathione: In human samples measured using 40 M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time.

• Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar manner to H2O2 so care must be used. DAF is a specific probe available for Nitric Oxide


Hydroethidine

HE EB

NCH2CH3

NH2H2N

H Br-NCH2CH3

NH2H2N

+

O2-

Phagocytic Vacuole

SODH2O2

NADPH

NADP

O2

NADPH Oxidase

OH-

O2-

DCFDCF

HE

OO22--

HH22OO22

DCFDCF

Example: Neutrophil Oxidative Burst


DCFH-DA DCFH DCF

COOHH

Cl

O

O-C-CH3

O

CH3-C-O

Cl

O

COOHH

Cl

OHHO

Cl

O

COOHH

Cl

OHO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’-dichlorofluorescin

2’,7’-dichlorofluorescin diacetate

2’,7’-dichlorofluorescein

Cellular Esterases

H2O2

DCFH-DA

DCFH-DADCFH-DA

DCFHDCFH

DCF

H OH O 2 22 2Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence.1

1000

100 10

1

0

20

40

60

cou

nts

PMA-stimulated PMNControl

80


Hydroethidine - Superoxide Production

15 minutes 45 minutes


Endothelial Cell Oxidative Pathways

Relative percentages of the mean intracellular EB fluorescence (O2

-) in rat pulmonary endothelial cells (REC) 60 min after stimulation with H2O2. This figure is a summary of a number of possible oxidative pathways in REC. The Y axis shows a measurement of superoxide anion via oxidation of hydroethidine to ethidium bromide, as a percentage of the control (100%). XO mediated pathways are inhibited by nearly 50%. A combination of inhibitors of mitochondrial respiration, as well as solvents indicate the baseline oxidation of the probe (30-40%). (n=3)

100

52.5 58.9

94.3

57.7

80.9

124.4118.9

50.241.5 42.4 44.6

34.3

0

20

40

60

80

100

120

140

aa

be

c

b b

dd

e.g.

f f fg

h

Mea

n C

han

nel

EB

Flu

ore

scen

ceP

erce

nta

ge

Ch

ang

e in

EC only

EC + 5mM A

llopurin

ol & 1mM

DM

SO

ECEC + 1mM

cyan

ide & 1mM

DM

SO

EC + 1mM C

yanide

EC + 1mM A

zide

EC + 1mM D

eferoxam

ine

EC + 10mM M

annito

l

EC + 8mM B

enzo

ate

EC + 1mM D

MSO

EC + 5mM D

PI

+ 5mM D

PI

EC + 5mM O

xypurinol

EC + 5mM A

llopurin

ol

+ 5mM A

llopurin

ol

EC + 5mM A

llopurin

ol & 1mM

Cn


Oxidative Reactions

• Superoxide Hydroethidine• Hydrogen Peroxide Dichlorofluorescein• Glutathione levels Monobromobimane• Nitric Oxide Dichlorofluorescein


Calcium Flux

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Rat

io: in

tens

ity o

f 46

0nm

/ 4

05nm

sig

nals

Time (seconds)

Time (Seconds)0 36 72 108 144 180

RAT

IO [s

hort/

long

]0

200

400

600

800

1000

StimulationStimulation

Flow Cytometry Image Cytometry


Membrane Potential

• Oxonol Probes • Cyanine ProbesHow the assay works:• Carbocyanine dyes released into the surrounding media as cells depolarize

• Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs

Time (sec)

Gre

en F

luor

esce

nce

Repolarized Cells

051

210

24

0 300 1500 1200 2400Time (sec)

051

210

24G

reen

Flu

ores

cenc

e

PMA Added fMLP Added

Depolarized Cells


Membrane Polarization

• Polarization/fluidity Diphenylhexatriene

How the assay works: The DPH partitions into liphophilic portions of the cell and is excited by a polarized UV light source. Polarized emissions are collected and changes can be observed kinetically as cells are activated.

An image showing DPH fluorescence in cultured endothelial cells.


CD16 Expression on Normal Cultured PMN

0 Hours

negativecontrol

48 Hours

24 Hours

As cells age, the CD16 expression reducesThe “bright” CD16 antigen is lost first


PI - Cell Viability

How the assay works:• PI cannot normally cross the cell membrane• If the PI penetrates the cell membrane, it is assumed

to be damaged• Cells that are brightly fluorescent with the PI are

damaged or dead

PIPI

PIPI

PIPI

PIPI

PIPI

PIPI

PIPI

PIPIPIPI

PIPI

PI

PIPI

PIPI

PIPI

Viable Cell Damaged Cell


Superoxide measured with hydroethidine

Step 7C: Export data from Excel data base to Delta Graph

Step 6C: Export data from measured regions to Microsoft Excel

cell 1

cell 2

cell 3cell 4

cell 5

Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.

-2000

200

400600800

10001200140016001800

cell 1

cell 2

cell 3

cell 4

cell 5

Time in seconds1000 1200 1400 1600 1800600 800 200 400%

chan

ge (

DC

F fl

uore

scen

ce)


Phagosome

O2

O2-

H2O2

NADPH + H+

NADP+

HMP

NADPH

Oxidase

GSSG

GSH

GRGP

H2O2SOD

O2-

H+

H2O

Catalase

H2O + O2

PCB

SOD

PCB(Reduced GSH level)

Stimulant

PKC

PCB

(PMA)

Human Neutrophil

?

?+

O2-

OH.

Lipid Peroxidation

Phospolipase A2 activity

Leukotrienes

H2O2


Ionic Flux Determinations

• Calcium Indo-1

• Intracellular pH BCECFHow the assay works:

• Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner

• The emission wavelength decreases as the probe binds available calcium

Time (Seconds)0 36 72 108 144 180

RAT

IO [s

hort/

long

]0

200

400

600

800

1000

StimulationStimulation0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Rat

io: i

nten

sity

of 4

60nm

/ 40

5nm

sig

nals

Time (seconds)

Flow Cytometry Image Analysis


Light Scatter Changes of PMN at 24 Hours

control

lps bu

ar


Phagocytosis• Uptake of Fluorescent labeled particles• Determination of intracellular or extracellular state of

particles

How the assay works:• Particles or cells are labeled with a fluorescent probe

• The cells and particles are mixed so phagocytosis takes place

• The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles

• The remaining fluorescence

represents internal particles

FITC-Labeled Bacteria


1

2

33

2

1

405/35 nm460 nm

Calcium ratioing study with Indo-1

Changes in the fluorescence were measured using the Bio-Rad calcium ratioing software. The same region in each wave length was measured and the relative change in each region

was recorded and exported to a spread sheet for analysis..

Export data from measured regions to Microsoft Excel

Export data from Excel data base to Delta Graph

50 100 150 2000

0.1

0.20.3

0.40.5

0.60.7

0.8

0

cell 1 cell 2 cell 3

Ratio: intensity1 (460nm) / intensity2 (405/35nm)

http://www.cyto.purdue.edu


On Calcofluor White

• No warranties on this one but Calcofluor White M2R (Fluorescent brightener28, Sigma) may work but staining may not be very specific. Lectins are another possible alternative.

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starchgranule distribution in cells of wheat (Triticum aestivum L.)caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca.11 microns in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells

Source:From: Richard Haugland ([email protected])Date: Thu Feb 07 2002 - 17:04:48 EST http://www.cyto.purdue.edu/hmarchiv/current/1041.htm

References: Biotech Histochem 1992 Mar;67(2):88-97Block-surface staining for differentiation of starch and cell walls in wheat endosperm. Glenn GM, Pitts MJ, Liao K, Irving DW. Western Regional Research Center, USDA-ARS, Albany, California 94710.


About SNARF-1

• SNARF®-1 carboxylic acid, acetate, succinimidyl ester http://www.probes.com/servlets/product?region=Select&item=22801 is a very new probe that we have not yet tested for assessing cell cycle.

• We have tested it for labeling cells and for cell tracing. I am not aware of any publications that have used it for that, however. However, it requires the same hydrolysis of the acetates as does CFSE and has the same succinimidyl ester as CFSE and should therefore have similar utility and have its fluorescence decrease by half on cell division, as does CFSE.

• Its potential advantage is that it can be excited at 488 nm but has red fluorescence so it may be complementary to CFSE.

Source: From: Richard Haugland ([email protected])Date: Thu Jan 17 2002 - 20:10:07 EST http://www.cyto.purdue.edu/hmarchiv/current/0872.htm

©1990-2013 J.Paul Robinson, Purdue University

Summary

• There are a variety of functional probes useful in flow cytometry

• Many require live cells for the entire assay period

• Timing for kinetic assays is critical

• You must match the probe to the excitation as usual

09:36 PM

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