A

rtvaeauwp©

K

1

eAcamca2iaa1

t

0d

Journal of Neuroscience Methods 161 (2007) 1–10

A rapid method to measure beta-amyloidinduced neurotoxicity in vitro

Dhara Patel 1, Theresa Good ∗Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County,

1000 Hilltop Circle, Baltimore, MD 21250, USA

Received 27 August 2005; received in revised form 2 August 2006; accepted 2 October 2006

bstract

�-Amyloid (A�) is the primary protein component of senile plaques in Alzheimer’s disease (AD) and is believed to be associated with neu-otoxicity in the disease. A�-induced neurotoxicity is strongly dependent on its structure. The aggregation of the peptide from monomeric formo fibrils is a function of many variables including time. It is because of this dynamic nature of A� structure that there is a necessity for an initro toxicity assay that is rapid enough to eliminate or at least lower the possibility of A� structural changes during the time required for thessay. Here we describe a fast and sensitive method with which to assess A�-induced neurotoxicity in SH-SY5Y neuroblastoma cells. The methodmploys two-color flow cytometry using annexin-phycoerythrin, a marker for cell surface phosphatidylserine that typically indicates early stages ofpoptosis, and 7-amino-actinomycin D, a membrane impermeant nucleic acid dye. We compare results using the two-color assay to those obtained

sing the propidium iodide toxicity assay and demonstrate comparability of results, but in 2 h or less with the two-color assay as opposed to 24–48 hith the propidium iodide assay. The assay described could be a useful tool in evaluating the role of A� structure in biological activity of theeptide. 2006 Elsevier B.V. All rights reserved.

xin; 7

os2

fc(a(mae

eywords: Alzheimer’s disease; �-Amyloid; SH-SY5Y; Flow cytometry; Anne

. Introduction

Alzheimer’s disease (AD) is the leading cause of neurodegen-ration in the United States, affecting approximately 4.5 millionmericans in 2003 (Hebert et al., 2003), with an annual cost of

are for these individuals estimated at over $100 billion (Ernstnd Hay, 1994; Koppel, 2003). One of the pathological hall-arks of AD is the formation of amyloid plaques in the cerebral

ortex, the primary protein component of which is the 39–43mino acid peptide �-amyloid (A�) (Gorman and Chakrabartty,001; Selkoe, 1994). There is a plethora of genetic, physiolog-cal and biochemical evidence that supports the idea that A� is

t least one of the originating causes of the disease (Batemannd Chakrabartty, 2004; Loo et al., 1993; Ross, 2004; Selkoe,999). A� monomers are thought to aggregate to form a variety

∗ Corresponding author. Tel.: +1 410 455 3403/5; fax: +1 410 455 1049.E-mail addresses: dhara1@umbc.edu (D. Patel),

good@umbc.edu (T. Good).1 Tel.: +1 410 455 3400; fax: +1 410 455 1049.

Smtp2etht

165-0270/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jneumeth.2006.10.004

-Aminoactinomycin D

f oligomeric and fibril species. The presence of aggregated A�pecies correlates with neurotoxicity (Gorman and Chakrabartty,001; Simmons et al., 1994).

The A� aggregation process, including the presence of dif-erent A� oligomers or aggregation states and their relativeoncentrations, depends on a number of factors, such as pHGarzon-Rodriguez et al., 1997; Schladitz et al., 1999), temper-ture, peptide concentration, type of solvent and pretreatmentFezoui et al., 2000; Nichols et al., 2005), presence of salt, theixing protocol (Burdick and Glabe, 1994; Walsh et al., 1997),

nd most important to this study, the time of aggregation orxposure to solvent (Fezoui et al., 2000; Nichols et al., 2005;chladitz et al., 1999; Tang and Zhang, 2001). There has beenuch interest over the past decade in identifying the structure of

oxic A� species such that therapeutics targeting these species orreventing their formation could be developed (De Felice et al.,004; Garzon-Rodriguez et al., 1997; Kayed et al., 2003; Walsh

t al., 1999; Wang et al., 2002a; Ward et al., 2000). Progressowards this goal of identifying the structure of toxic A� speciesas been hampered because of the dynamic nature of A� struc-ure and the difficulty of ensuring that the structure of A� that

2 urosci

wic

sSai(cAitvditeacre1tmht

uaamr1tmawgdP

rt

2

2

(ratGMAirM

2

(irttpab(pwago

Fwb

D. Patel, T. Good / Journal of Ne

as added to cells in culture at the beginning of a toxicity exper-ment is the same as the structure of A� that interacted with theell resulting in toxicity.

Conventional methods of assessing the neurotoxicity of A�amples such as the MTT reduction assay (Loo et al., 1993;hearman et al., 1994), lactate dehydrogenase assay (Fezoui etl., 2000), trypan blue assay (DeFelice et al., 2001), the propid-um iodide (PI) assay (Martin et al., 2001) and TUNEL assayColurso et al., 2003) require incubation of A� samples withells for 24 h or more, during which time the structure of the� species is likely to change. This change results in ambigu-

ty in identifying the structural species and the concentrationshereof that are responsible for the neurotoxic effects observedia these assays. One possible solution to this problem is toesign a toxicity assay that requires the least possible A�-cellncubation time. To this end, efforts have been made in the pasto use other cellular responses as indicators of eventual A� toxicffect. These indicator responses include A�-induced G-proteinctivation (Rymer and Good, 2001; Wang et al., 2003), acetyl-holinesterase expression (Zhang et al., 2003), oxidative stressesponses (Shearman et al., 1994; Varadarajan et al., 2000; Whitet al., 1998), changes in ion channel function (Good and Murphy,996; Weiss et al., 1994) and changes in cell membrane poten-ial (Blanchard et al., 2002; Bobich et al., 2004). While these

ethods are more rapid than the traditional toxicity assays, it isard to correlate those cellular responses directly to an eventualoxic response.

Here we describe the use of a rapid apoptosis assaysing annexin V-phycoerythrin (annexin-PE) and 7-amino-ctinomycin D (7-AAD), a phosphatidylserine binding proteinnd a membrane impermeant nucleic acid dye, respectively, toeasure A�-induced neurotoxicity in SH-SY5Y human neu-

oblastoma cells. Toxic effects of A� can be seen in as little as5 min with the assay, and after 2 h, results are comparable withhose observed after 24–48 h with a classic PI assay. Further-

ore, we demonstrate that for A� samples that are allowed toggregate for various periods of time, with the two-color assay

e were able to elucidate differences in toxicity of certain aggre-ation intermediate samples of A�, while we were unable toetect such differences in viability of A� treated cells using theI assay. The use of the two-color assay may be useful in the

2

a

ig. 1. Representative flow cytometry histogram of PI stained SH-SY5Y neuroblastoith mixing (b). Gate P1 shows the viable population that is relatively unstained byrightly stained (dead cell) population increases.

ence Methods 161 (2007) 1–10

apid detection of A� induced changes in cells, especially whenransient structures of A� are being examined.

. Materials and methods

.1. Materials

A�(1–40) was purchased from Biosource InternationalCamarillo, CA) and Anaspec (San Jose, CA). Human neu-oblastoma SH-SY5Y cells were purchased from ATCC (Man-ssas, VA). Minimum essential media (MEM), other cell cul-ure reagents and cell dissociation buffer were purchased fromibco-Invitrogen (Grand Island, NY). PI was purchased fromolecular Probes (Eugene, OR). Annexin-PE, 7AAD andnnexin V binding buffer were purchased from Becton Dick-

nson Biosciences Pharminogen (San Diego, CA). All othereagents were purchased from Sigma Chemical Co. (St. Louis,

O).

.2. Peptide preparation

Stock solutions of A�(1–40) were prepared as describedRymer and Good, 2001). One milligram of A� was dissolvedn 100 �l of dimethyl sulfoxide (DMSO) or 0.1% (v/v) trifluo-oacetic acid in water. The peptide was incubated until dissolved,hen peptide stock solutions were diluted to desired concentra-ions in sterile MEM when used for viability assays or 10 mMhosphate buffered saline (PBS) when used for Congo Redssays. Samples were either rotated for 24 h at 11 rpm and 25 ◦Cefore use when predominately fibril aggregates were desiredFigs. 1–7), or samples were kept stationary at 37 ◦C for variouseriods of time when different A� aggregation intermediatesere desired (Fig. 8). A� was always added to cells exactly as

ggregated and without dilution, to ensure that what ever aggre-ated species formed during the aggregation period (0, 4, 8, 24r 72 h) were not altered by dilution into culture medium.

.3. Cell culture

Human neuroblastoma SH-SY5Y cells were cultured inhumidified 5% CO2/air incubator at 37 ◦C in MEM,

ma treated with no A� (a) and with 100 �M A� that was aggregated for 24 hPI. With A� treatment, the percentage of viable cells decreases and that of the

D. Patel, T. Good / Journal of Neuroscience Methods 161 (2007) 1–10 3

Fig. 2. Normalized viability of SH-SY5Y cells as a function of time of incu-bation with 100 �M A� as assessed by the PI assay. A� was aggregated for24 h with mixing prior to addition to cells. Mean viability measurements plusop

sN2wtf

2

1CrMdPw1aapwlawtwi

2

ciiiC7t

Fig. 3. Representative histograms of 7AAD staining (left panel) and dot-plotsof annexin-PE and 7AAD staining (right panel) of untreated cells (a, f), cellsta

2fB

r minus their standard deviation (error bars) are shown for three or more inde-endent measurements.

upplemented with 10% (v/v) fetal bovine serum, 2.2 mg/mlaHCO3, 100 �g/ml streptomycin, 100 U/ml penicillin, and.5 �g/ml Fungizone (amphotericin B). For the toxicity assaysith intermediate A� structural species, the cells were differen-

iated using 20 ng/ml solution of nerve growth factor-� in MEMor 5–7 days prior to treatment with peptide solutions.

.4. PI assay

SH-SY5Y cells were seeded in 96-well plates at a density of05 cells/well. After 24 h incubation of cells in a humidified 5%O2/air incubator and 37 ◦C (or 5–7 days for differentiated neu-

oblastoma), culture medium was replaced with 100 �l of A� inEM. Cells were incubated with A� at the same culture con-

itions for desired intervals of time. Just prior to staining withI to assess neurotoxicity, medium in the wells was replacedith 150 �l of dissociation buffer and incubated at 25 ◦C for5 min. Ten microliters of 33 �M PI was added to each wellnd the plate was left to incubate at 25 ◦C for 20 min in the darkfter brief shaking. Cells were mechanically scraped using sterilelastic loops. Immediately after cell staining, cell fluorescenceas measured using flow cytometry (BD FACSArray Bioana-

yzer, San Diego, CA). The cells were excited with a 532 nm lasernd fluorescence was detected using a 564–606 nm filter. Gatingas done so as to obtain percentages of the total cell population

hat were viable (unstained with PI). Normalized cell viabilityas estimated by dividing the percentage of viable population

n the sample by that in the control samples without A�.

.5. Annexin–7AAD Assay

Cells were seeded in 96-well plates and incubated underulture conditions as done for the PI assay. To evaluate the tox-city of the samples after incubation with A� for desired timentervals, the medium was replaced with 100 �l of 1× bind-

ng buffer (0.01 M Hepes/NaOH, pH 7.4, 0.14 M NaCl, 2.5 mMaCl2). Thereafter, 5 �l of both annexin-PE (5 ng/well) andAAD (0.25 �g/well) were added, followed by 10 min incuba-ion in the dark at 25 ◦C. One Hundred and fifty microliters of

e((l

reated with 100 �M A� for various periods of time (15 min (b, g), 1 h (c, h),nd 8 h (d, i)), and hydrogen peroxide treated positive controls (e, j).

× binding buffer was then added. Finally, cells were removedrom plates by mechanical scraping, then analyzed using theD FACSArray Bioanalyzer flow cytometer. Annexin-PE was

xcited with the 532 nm laser and detected using the yellow564–606 nm) filter, while 7AAD was detected using the red653–669 nm) filter. Spillover for the two detectors was calcu-ated using three samples one of which was unstained, another

4 D. Patel, T. Good / Journal of Neuroscience Methods 161 (2007) 1–10

Fig. 4. (A) Median 7AAD fluorescence intensity of samples treated with A�

for different time periods (�) (all data points significantly different from 0 timepoint (p < 0.05)); median of brightly stained peak (�) and unstained peak in con-trol samples (�). No statistically significant change in control peak fluorescenceintensity at any time was observed. The average of the median fluorescenceintensity was plotted for three or more independent measurements. Error barsrepresent the standard deviation of the means. (B) Representative 7AAD his-tograms with deconvolution results shown. Medians of the live, leaky and dead

Fig. 5. Extended �-sheet content of A� as a function of total A� concentrationas obtained from the Congo Red binding assay. Total concentration of fibrillarA� (�) and fractional fibrillar A� (�) are plotted. Fractional fibrillar A� wascalculated as the total fibrillar A� concentration divided by the total A� con-centration. A� samples were aggregated for 24 h with mixing in PBS prior toCongo Red assay. The mean of three measurements is presented with error barsrepresenting the standard deviation of the mean.

Fig. 6. Normalized viability of SH-SY5Y cells as a function of A� concentrationap(

s7iMwwu

cllcswuEtiaopta

s assessed by the PI assay. A� samples were aggregated for 24 h with mixingrior to addition to cells. Cells were incubated with A� for 24 h (�) and 48 h�).

tained with only annexin-PE and the third stained only withAAD. To ensure staining, positive controls were prepared byncubating cells with a 533 �M H2O2 solution in 33% (v/v)

EM in deionized water (Wang et al., 2002b). A quadrant gate

as used to estimate the percentage of the total population thatas both annexin and 7AAD negative, taken as the viable pop-lation.

ell populations were fixed at channel 105, 145, and 175, corresponding theow, medium and high fluorescence intensities of 7AAD. The histogram on theeft is of a control population, while the histogram on the right is of A� treatedells. (C) Percentages of live (�), leaky (�), dead (�) and (leaky + dead) (�)ubpopulations of cells treated with 100 �M A� for varying time periods. A�

as aggregated for 24 h with mixing prior to addition to cells. Percent cell pop-lations were estimated by deconvolution of 7AAD histograms into three peaks.rror bars represent uncertainty in deconvolution of at least three different his-

ograms. (D) Normalized viability of SH-SY5Y cells as a function of time ofncubation of cells with 100 �M A�, as assessed by the Annexin-PE and 7AADssay. Viability was estimated as the fraction of cells in the lower left quadrantf the annexin–7AAD dot-plot, with gates set for positive and negative controlopulations. The mean viability of three or more measurements is plotted. Sta-istically significant changes in cell viability were detected at all times measuredfter 0 time (p < 0.05).

D. Patel, T. Good / Journal of Neuroscience Methods 161 (2007) 1–10 5

Fig. 7. (A) Normalized viability of SH-SY5Y cells assessed by the deconvolu-tion of 7AAD histograms as a function of A� concentration. A� samples wereaggregated for 24 h with mixing prior to addition to cells. Cells were incubatedwith A� for 2 h. Percentages of live (�), leaky (�), and dead (�) subpopulationsof cells. (B) Normalized viability of SH-SY5Ycells assessed by the annexin-PEaaw

2

7mttutow7c

2

oCotwa

Fig. 8. (A) Extended �-sheet content of A� peptide samples that have been incu-bated at 37 ◦C under quiescent conditions for various periods of time as obtainedfrom the Congo Red binding assay. The values for the 4 and 8 h intermediate A�

species are statistically different from the 72 h fibrillar sample (p < 0.05). The 8 hintermediate and 72 h fibrillar samples have statistically higher �-sheet contentcompared to the fresh peptide sample (p < 0.05). (B) Normalized viability ofA� species as assessed by PI assay. Hundred micromolars A� was incubated at37 ◦C without mixing for various periods of time (0, 4, 8, and 72 h) prior to addi-tion to cells. Cells were exposed to A� for 24 h prior to staining with PI. Onlycell viability associated with the A� sample that was aggregated for 72 h priorto cell addition was statistically different from that of the fresh peptide sample(p < 0.05). (C) Normalized viability of A� species incubated at 37 ◦C withoutmixing for various periods of time as obtained from the annexin-PE and 7AADtwo-color assay. Hundred micromolars A� was used. Peptide was aggregatedfor 0, 4, 8 and 72 h in MEM prior to addition to cells without further dilutionof peptide. Cells were exposed to A� for 2 h prior to staining with annexin-PEand 7AAD. Cell viability associated with A� species preincubated for 4 and 8 h

nd 7AAD two-color assay as a function of A� concentration. A� samples wereggregated for 24 h with mixing prior to addition to cells. Cells were incubatedith A� for 2 h.

.6. Deconvolution of 7AAD histograms

Flow cytometry data obtained from the annexin-PE andAAD two-color assay was analyzed in several ways to esti-ate the percentage of live population in the samples. One of

he methods was to deconvolute the 7AAD log histogram intohree subpopulations, i.e. live, leaky and dead. This was donesing the ‘proliferation wizard’ tool in ModFit LT 3.1. The cen-ers of the live, leaky and dead population were fixed. The shapesf each population were assumed to be log-normal. The softwareas used to perform a non-linear least squares regression of theAAD histograms such that the percentage of each populationould be estimated.

.7. Congo Red staining

Congo Red studies were performed to assess the presencef amyloid fibrils or extended �-sheet content in A� solution.ongo Red dye was dissolved in PBS to a final concentration

f 120 �M. Congo Red solution was added to the peptide solu-ions at the ratio of 1:9. The peptide solution and control solutionere allowed to interact with Congo Red for 30–40 min prior to

bsorbance measurement with a Model 420 UV–vis spectropho-

prior to addition to cells were statistically different from the viability associatedwith fresh and the 72 h fibrillar samples (p < 0.05).

6 urosci

tfu

[

w4

wFrt

2

pfic

3

3w

utsflw(wh

riFnsocv

csccsAtdapt

tradAubahai

vAliht7liwqlcrotrwts

mTosfloow

ipdohlasfTA

D. Patel, T. Good / Journal of Ne

ometer (Spectral Instruments, Tucson, AZ) at 25 ◦C. The fibrilormation of the samples was estimated from the absorbancesing the following equation:

A�Fib] =(

541At

4780

)−( 403At

6830

)−( 403ACR

8620

)(1)

here [A�Fib] is the concentration of A� fibril, 541At, 403At and03ACR are the absorbances of the sample and Congo Red at theavelength of 541 and 403 nm, respectively (Klunk et al., 1999).rom these data, fractional fibril content was calculated as theatio of sample fibril concentration to total A� concentration inhe sample.

.8. Statistical methods

The Student’s two-tailed t-test or the two-way ANOVA singlearameter post hoc analysis was used to analyze statistical dif-erences between data. Significance was taken as p < 0.05 unlessndicated. In all cases 3 or more independent measurements ofell viability were made.

. Results

.1. SH-SY5Y viability as a function of time when treatedith Aβ, a comparison of two viability assays

We sought to establish comparability between a commonlysed cell viability assay and a less commonly used early apop-osis assay in a cell culture model of A� toxicity. In Fig. 1, wehow representative histograms obtained with PI staining andow cytometry of SH-SY5Y human neuroblastoma cells treatedith 100 �M A� in fibril form for 24 h. Untreated control cells

Fig. 1a) have a larger percentage of unstained or viable cells,hile those treated with 100 �M A� have a larger percentage ofighly fluorescent or dead cells.

We then examined the time of A� incubation with cellsequired such that a significant toxic response to 100 �M A�n fibril form could be detected using the PI assay. As seen inig. 2, loss of cell viability with A� treatment was more pro-ounced with time. ANOVA post hoc analysis indicated thatignificant loss of viability compared to untreated controls wasbserved only after 12 or more hours of A� incubation withells (p < 0.05). There was no statistically significant change iniability between 24 and 48 h incubation of cells with A�.

We performed analogous experiments, incubating SH-SY5Yells for various lengths of time with fibril A� and then mea-ured viability using an annexin-PE–7AAD early apoptosis flowytometry assay. Representative histograms of 7AAD fluores-ence intensity and of dot-plots of annexin-PE versus 7-AADtaining are shown in Fig. 3. Negative controls, cells treated with� for 15 min, 1 h, 8 h, and positive controls are shown. From

he 7AAD flow cytometry histograms seen in Fig. 3A–E, three

ifferent populations of cells can be seen, with high, medium,nd low intensity 7AAD staining, corresponding to dead cells,artially permeable cells, and live cells. In the untreated nega-ive controls (Fig. 3A), the two predominant peaks correspond

emfe

ence Methods 161 (2007) 1–10

o the brightly stained and unstained, dead and live populations,espectively. In the peroxide treated positive controls (Fig. 3E),gain, the most prominent peaks correspond to brightly stainedead and unstained live cell populations. However, for all of the� treated cells, even at times as short as 15 min, a new cell pop-lation was seen, whose fluorescence intensity was intermediateetween the live and dead cell populations identified in positivend negative controls (Fig. 3B–D). Although not shown here,istograms of negative control cells and cells treated with A�nd then immediately subjected to flow cytometry analysis (zeroncubation time) appeared identical.

Fig. 3F–J shows representative dot-plots of annexin stainingersus 7AAD staining for negative controls, cells treated with� for 15 min, 1 h, 8 h, and positive controls. Cells in the lower

eft quadrant of each plot, with low annexin and 7AAD stain-ng, are considered viable, those in the upper left quadrant, withigh annexin, low 7AAD staining are considered early apoptotic,hose in the upper right quadrant, with high annexin and highAAD staining are considered late apoptotic and those in theower right quadrant, with low annexin and high 7AAD stain-ng are considered necrotic. In negative controls (Fig. 3F), thereere two predominant cell populations, one in the lower leftuadrant and one in the upper right quadrant, corresponding toive cells and late apoptotic cells, respectively. In the positiveontrols (Fig. 3J), the cell population has shifted to the upperight, apoptotic quadrant. In A� treated cells, the percentagef cells in the lower left or viable cell quadrant decreases whilehose in the lower and upper right (necrotic and apoptotic) quad-ants increases. The most obvious change in A� treated cellsas the increase in 7AAD staining (the cell population shifts

o the right), while there is relatively little increase in annexintaining.

We examined several measures of cell viability after A� treat-ent for the annexin-PE–7AAD stained cells, shown in Fig. 4.he change in median 7AAD fluorescence intensity as a functionf time of A� treatment (Fig. 4A), provides a qualitative mea-ure of the membrane permeability of the population. Medianuorescence intensity of A� treated cells increased at times up tone hour, then decreased slightly. Median fluorescence intensityf positive and negative control cells did not change significantlyith time.Because 7AAD staining of A� treated cells appeared to result

n three populations of cells, consistent with a live (unstained)opulation, a dead (highly stained) population, and an interme-iately stained population which we refer to as “leaky”, insteadf simply gating the 7AAD histograms, we deconvoluted theistograms to determine the percentage of cells in each popu-ation. In Fig. 4B, an example the results of the deconvolutionre shown, with the peaks associated with the three populationshown. Fig. 4C shows the percentages of these subpopulationsound in the samples treated with A� for varying periods of time.he percentage of live cells decreased after 15 min of exposure to�, while the percentage of leaky cells increased after the same

xposure time. The percentage of dead cells, as estimated via thisethod, did not change significantly at any time of exposure

rom 15 min to 8 h (p > 0.05). There was no statistical differ-nce in the percentages of the live, dead or leaky subpopulations

euros

ws

ta(ibsbdriiwcoea

3c

�slfiaafisicaoefia

oPicrwtoc(ocopAoA

3c

bbaiP

etsr7eOaau(

satotvwgsg

4

sadaribasdoalutAi

D. Patel, T. Good / Journal of N

ith incubation time for the untreated control cells (data nothown).

We also used the annexin-PE–7AAD dot-plots and estimatedhe fraction of viable cells, cells that had both low annexinnd low 7AAD staining using conventional quadrant gatingFig. 4D). In as little as 15 min, a statistically significant decreasen cell viability was observed (p < 0.05). Though there seems toe a local minima in viability around the 1 h time period, aaturation is reached by 2 h of incubation with A�, followedy no significant changes in 7AAD staining thereafter. Theecrease in viability of A� treated cells we estimated with quad-ant gating of the annexin–7AAD dot-plots after about 1 h ofncubation with fibril A�, corresponds to a similar decreasen the leaky population and increase in dead cell populatione identified from deconvolution of the 7AAD histograms. We

annot explain the local minima in viability of cells after 1 hf exposure to fibril A�, so chose to use another time of A�xposure (2 h) for further comparisons of the annexin–7AADssay.

.2. SH-SY5Y viability as a function Aβ concentration, aomparison of two viability assays

A�-induced neurotoxicity has been correlated to the extent of-sheet structure present in a sample of peptide. We prepared A�amples at various concentrations, aggregated them for 24 h, aength of time considered appropriate for formation of stable A�brils, then measured both extended �-sheet content of samplesnd cell response in two viability assays, the PI assay and thennexin-PE–7AAD assay. In Fig. 5, the extended �-sheet orbrillar content of the A� samples prepared for all the toxicitytudies, as determined from Congo Red binding experiments,s shown. At low A� concentrations, extended �-sheet or fibrilontent increased with concentration between 10 and 20 �M,t which concentrations a local maximum in fibril content wasbserved. When the data was normalized by the total A� inach sample (fractional fibril content), the local maximum inbril content at intermediate concentration ranges became morepparent.

The viability of SH-SY5Y cells upon incubation with vari-us concentrations of A� for 24 and 48 h as determined via theI assay are shown in Fig. 6. As the concentration of A� was

ncreased, cell viability decreased, but with a local minimum inell viability at 10–20 �M A�. Analogous experiments were car-ied out using the annexin-PE and 7AAD assay, except that cellsere incubated with various concentrations of A� for 2 h prior

o measurement, and viability was estimated via deconvolutionf the 7AAD histograms (Fig. 7A) and from the population ofells in the lower left quadrant of annexin-PE–7AAD dot-plotsFig. 7B). As seen when using the PI assay, with 7AAD aloner with the annexin-PE–7AAD assay, cell viability drops at lowoncentrations of A�, and a local minimum in viability wasbserved at 10–20 �M A�. Also of note, as seen in Fig. 7A, the

opulation of leaky cells was increased at high concentrations of� compared to that at 20 �M A�, while it was the populationf dead cells that was elevated at 20 �M A� compared to other� concentrations.

ru

a

cience Methods 161 (2007) 1–10 7

.3. SH-SY5Y viability for intermediate species of Aβ, aomparison of two viability assays

To examine if the two-color annexin-PE–7AAD assay coulde used to detect changes in A� toxicity associated with unsta-le and/or intermediate A� species, we prepared A� samplesggregated for different times, then examined Congo Red bind-ng and viability of cells treated with A� samples using both theI and two-color assay.

Fig. 8A shows the extended �-sheet content of the differ-nt A� samples allowed to aggregate for varying periods ofime at 37 ◦C. The 4 and 8 h intermediate A� species containedtatistically different �-sheet content compared to the 72 h fib-illar sample (p < 0.05). Moreover, the 8 h intermediate and the2 h fibrillar A� samples contain a statistically higher content ofxtended �-sheet compared to the fresh A� sample (p < 0.05).nly the 72 h aggregated A� sample contained obvious fibrils

s measured using electron microscopy (data not shown). A�ggregated under static conditions for 4 and 8 h contained sol-ble low molecular weight aggregates as determined via FPLCdata not shown).

Fig. 8B and C show the viability of cells treated with A�amples that had been aggregated for various lengths of times determined using the PI and annexin–7AAD assays, respec-ively. When viability was measured using the PI assay (Fig. 8B),nly the 72 h fibrillar samples were statistically more toxic thanhe fresh A� samples. However, when using the annexin–7AADiability assay all of the aggregated samples (i.e. 4, 8 and 72 h)ere statistically more toxic to cells than the fresh unaggre-ated sample of A�. Moreover, the A� aggregation intermediateamples (4 and 8 h preincubation of peptide) had a statisticallyreater toxicity than the fibrillar 72 h sample.

. Discussion

More and more studies today implicate relatively unstablemall oligomers or soluble aggregates of A� as the speciesssociated with the neurotoxic effects observed in Alzheimer’sisease (Chromy et al., 2003; Gibson et al., 2004; Kayed etl., 2003; Walsh et al., 2002). In order to better understand theelationship between A� structure and toxic response, it wasmportant to develop a toxicity assay such that the time of via-ility measurement was shorter than or comparable to the timessociated with structure change of A� species. In vitro, conver-ion of A� monomer to dimer occurs within minutes or faster,imer to larger oligomer occurs within minutes to hours, andligomer to protofibrils or fibril occurs within hours (Pallittond Murphy, 2001). While a variety of factors such as peptideength, temperature, concentration, and buffer/solvent can besed to slow changes in A� structure, some of these manipula-ions cannot be used in the presence of viable cells (Gursky andleshkov, 2000; Williams et al., 2005). We thus set out to exam-

ne different viability assays and establish the comparability of a

apid apoptosis assay with a conventional in vitro toxicity assaysed with A�.

In the past, the combination of annexin and PI has been useds an early measure of apoptosis in in vitro assays (Sennvik

8 urosci

efbsiaaoaoomeT

Aecaaouiti2Aearffiu

pwtmmPtdaeFtcaaitile7

a

i(t1cTab2btoab7paAi

ecitudvvat

abhAwtWttiTwabtoftat(2

D. Patel, T. Good / Journal of Ne

t al., 2001; Tong et al., 2004). Annexin V has a high affinityor phosphatidylserine (PS) (Raynal and Pollard, 1994) and cane used to detect changes in PS on the outer cell membraneurface that occur early in apoptosis, prior to loss of membranentegrity (Fadok et al., 1992; Homburg et al., 1995; Koopman etl., 1994). PI is a nucleic acid dye that is membrane imperme-nt and thus can be used to detect cells that are either necroticr in the later stages of apoptosis. Two-color flow cytometryssays utilizing annexin V bound to a fluorescent dye and PI orther membrane impermeant dyes have been used by a numberf researchers to detect early apoptotic changes in cell cultureodels of neurotoxicity and/or neurodegeneration (O’Donovan

t al., 2001; Schmitz and Friedrich, 2002; Shutte et al., 1998;riyoso and Good, 1999).

In this work, we show the comparability of results of in vitro� toxicity assays using PI and a two-color flow cytometry assay

mploying annexin-PE and 7AAD. In Figs. 1–4, we show thathanges in viability in SH-SY5Y cells could be seen in as littles 15 min after incubation with aggregated 100 �M A� using thennexin-PE–7AAD assay or 7AAD staining alone, while 12 hr more were needed to observe significant changes in viabilitysing the PI assay. In Figs. 6 and 7, we show that similar changesn viability could be measured in SH-SY5Y cells when exposedo different concentrations of aggregated A�, when cells werencubated with A� for 24–48 h using the PI assay (Fig. 6) orh using the 7AAD assay or annexin-PE–7AAD assay (Fig. 7).t all concentrations of A� but 100 �M, the PI assay after 48 h

xposure to A�, and the 7AAD assay and annexin–7AAD assayfter 2 h exposure to A� gave quantitatively similar viabilityesults. In all of these samples A� was aggregated with mixingor 24 h such that structure would be predominately fibril (con-rmed by electron microscopy, data not shown) and relativelynchanging.

When using the annexin-PE–7AAD assay, we noted that aopulation of cells incubated with 100 �M A� then stainedith 7AAD exhibited staining intermediate between live (nega-

ive) and dead (positive) controls (Fig. 3). These cells had someembrane permeability to 7AAD. Similar intermediate levels ofembrane permeability were not observed when staining withI (Fig. 1). 7AAD has been shown by others to pass through

he cell membrane more quickly than PI and has been used toetect unstained or live cells, dimly stained or early apoptotic,nd highly stained or late apoptotic or necrotic cells (Philpottt al., 1996). The intermediate stained cell population seen inig. 3, after treatment with A�, may represent an early apop-

otic cell population, or may possibly indicate changes in theell associated with A� induced increased membrane perme-bility (Ambroggio et al., 2005; Demuro et al., 2005; Kayed etl., 2004; McLaurin et al., 1998). This can be seen most vividlyn Fig. 4C. The earliest change in cells associated the exposureo A� detected in this work was the increase in 7AAD stain-ng to intermediate levels that we interpret to be associated witheaky cells (Fig. 4C). The population of dead cells detected upon

xposure to A� in the first 8 h was roughly equivalent, whetherAAD alone or PI alone was used to stain cells.

Results from both the PI assay and the annexin–7AADssay showed that A� toxicity to SH-SY5Y cells was a max-

2

Po

ence Methods 161 (2007) 1–10

mum at an intermediate concentration, around 10–20 �M A�Figs. 6 and 7). While somewhat unexpected, we also observedhat A� Congo Red binding had a local maximum at between0 and 20 �M A� (Fig. 5), indicating that there were signifi-ant differences in A� structures at the different concentrations.here is some precedent to indicate that A� aggregation changess a function of concentration, and that more toxic species cane formed at lower A� concentrations (Pallitto and Murphy,001). Alternatively, at low concentrations A� aggregation maye via a predominately fibril pathway, while at high concentra-ions, more amorphous aggregation may occur. In either case,ur results suggest that A� may act on cells via different mech-nisms at high and low concentrations. As seen in Fig. 7A,elow 50 �M A�, the percentage of dead cells detected viaAAD staining increased with A� concentration, while theercentage of leaky cells was relatively unchanging. However,bove 50 �M A�, the percentage of leaky cells increased with� concentration, while the percentage of dead cells did not

ncrease.We show that both annexin-PE–7AAD two-color flow cytom-

try and 7AAD staining alone can be used for rapid detection ofhanges in cells upon exposure to A�. When dual color stain-ng was used, simple quadrant gating was sufficient to estimatehe viable cell population while when only 7AAD staining wassed, deconvolution of the 7AAD histograms was necessary toetermine viable cell population. The performance of the decon-olution algorithm was somewhat dependent on the initial guessalues of the peak medians and their widths. However, with thedded analysis of the 7AAD histograms alone, added informa-ion about the cell response to A� was obtained.

We established comparability of using PI, 7AAD, ornnexin–7AAD in combination by using A� structures that hadeen aggregated for relatively long times and by most measuresad relatively stable fibril structures. However, when we used� samples that were aggregated for short times, structures thatould be expected to be non-fibril and less stable, we found that

he different assays did not yield comparable results (Fig. 8).hen using the two viability assays to detect changes in A�

oxicity as a function of aggregation time of A� prior to additiono cells, only with the annexin-PE–7AAD assay were changesn viability at intermediate aggregation times detected (Fig. 8).he standard PI assay that required 24 h exposure of peptideith cells could not be used to detect toxicity of non-fibril,

nd presumably unstable, A� aggregation intermediates. Weelieve this is because the A� aggregation intermediate struc-ures changed during the time course of the PI assay. In addition,nly when using the two-color annexin-PE–7AAD assay weound that intermediate aggregation times led to A� structureshat were more toxic than fibril containing samples. These resultsre consistent with the growing body of literature that suggestshat A� oligomers, not fibrils, are the more toxic A� speciesChromy et al., 2003; Gibson et al., 2004; Glabe and Kayed,006; Kayed et al., 2003; Townsend et al., 2006; Walsh et al.,

002).

In summary, we demonstrate the utility of an annexin-E–7AAD two-color flow cytometry assay for rapid detectionf cellular changes upon exposure to A�. The use of a rapid

euros

vu

A

(

R

A

B

B

B

B

C

C

D

D

D

E

F

F

G

G

G

G

G

G

H

H

K

K

K

K

K

L

M

M

N

O

P

P

R

R

R

S

S

S

S

D. Patel, T. Good / Journal of N

iability assay may be particularly useful when examiningnstable A� structures.

cknowledgement

This work was funded by the National Institutes of HealthNS042686 to TAG).

eferences

mbroggio E, Kim D, Separovic F, Barrow C, Barnham K, Bagatolli L, et al.Surface behavior and lipid interaction of Alzheimer beta amyloid peptide1–42: a membrane disrupting peptide. Biophys J 2005:2706–13.

ateman DA, Chakrabartty A. Interactions with Alzheimer amyloid peptideswith cultured cells and brain tissue, and their biological consequences.Biopolymers (Peptide Science) 2004;76:4–14.

lanchard B, Thomas V, Ingram V. Mechanism of membrane depolarizationcaused by the Alzheimer’s Abeta 1–42 peptide. Biochem Biophys Res Com-mun 2002;293:1197–203.

obich J, Zheng Q, Campbell A. Incubation of nerve endings with a physiolog-ical concentration of Abeta1–42 activates CaV2.2 (N type) voltage operatedcalcium channels and acutely increases glutamate and noradrenaline release.J Alzheimer’s Dis 2004;6:243–55.

urdick D, Glabe C. Assembly and aggregation properties of syn-thetic Alzheimer’s A4/beta amyloid peptide analogs. J Biol Chem1994;267:546–54.

hromy B, Nowak R, Lambert P, Viola K, Chang L, Velasco P, et al.Self assembly of Abeta (1–42) into globular neurotoxins. Biochemistry2003;42:12749–60.

olurso GJ, Nilson JE, Vervoort LG. Quantitable assessment of DNA fragmen-tation and beta-amyloid deposition in insular cortex and midfrontal gyrusfrom patients with Alzheimer’s disease. Life Sci 2003;73:1795–803.

e Felice FG, Vieira MN, Saraiva LM, Figueroa-Villar JD, Garcia-Abreu J,Liu R, Chang L, Klein WL, Ferreira ST. Targeting the neurotoxic speciesin Alzheimer’s disease: inhibitors of Abeta oligomerization. FASEB J2004;18:1366–72.

eFelice FG, Houzel JC, Garcia-Abreu J, Louzada PRF, Afonso RC, MeirellesMNL, et al. Inhibition of Alzheimer’s disease �-amyloid aggregation, neuro-toxicity and in vivo deposition by nitrophenols: implications for Alzheimer’stherapy. FASEB 2001;15:1297–9.

emuro A, Mina E, Kayed R, Milton S, Parker I, Glabe C. Calcium dysregulationand membrane disruption is ubiquitous neurotoxic mechanism of solubleamyloid oligomers. J Biol Chem 2005;280:17294–300.

rnst RL, Hay JW. The U.S. economic and social costs of Alzheimer’s diseaserevisited. Am J Public Health 1994;84:1261–4.

adok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM.Exposure of phosphatidylserine on the surface of apoptotic lymphocytestriggers recognition and removal by macrophages. J Immunol 1992;148:2207–16.

ezoui Y, Hartley DM, Harper JD, Khurana R, Walsh DM, Cordron MM, et al. Animproved method of preparing the amyloid-� protein for fibrillogenesis andneurotoxicity experiments. Amyloid: Int J Exp Clin Invest 2000;7:166–78.

arzon-Rodriguez W, Sepulveda-Beeerra M, Milton S, Glabe CG. Soluble amy-loid A�(1–40) exists as a stable dimer at low concentrations. J Biol Chem1997;272:21037–44.

ibson G, Gunasekera N, Lee M, Lalyveld V, El-Agnaf O, Wright A, et al.Oligomerization and neurotoxicity of the amyloid ADan peptide implicatedin familial Danish dementia. J Neurochem 2004;24:3801–9.

labe CG, Kayed R. Common structure and toxic function of amyloid oligomersimplies a common mechanism of pathogenesis. Neurology 2006;66:S74–8.

ood TA, Murphy RM. Effect of beta-amyloid block of the fast-inactivating K+

channel on intracellular Ca2+ and excitability in a modeled neuron. ProcNatl Acad Sci USA 1996;93:15130–5.

orman PM, Chakrabartty A. Alzheimer’s �-amyloid peptides: structures ofamyloid fibrils and alternate aggregation products. Biopolymers (PeptideScience) 2001;60:3881–93.

S

cience Methods 161 (2007) 1–10 9

ursky O, Aleshkov S. Temperature-dependent beta-sheet formationin beta-amyloid Abeta (1–40) peptide in water: uncoupling beta-structure folding from aggregation. Biochim Biophys Acta 2000;1476:93–102.

ebert LE, Scherr PA, Bienias JL, Bennett DA, Evans DA. Alzheimer diseasein the U.S. Arch Neurol 2003;60:1119–22.

omburg CH, de Haas M, von den Borne AK, Verhoeven AJ, ReutelingspergerCPM, Roos D. Human neutrophils lose their surface Fc gamma RIIIand acquire annexin V binding sites during apoptosis in vitro. Blood1995;85:532–40.

ayed R, Head E, Thompson J, McIntire TM, Milton S, Cotman CW, et al. Com-mon structure of soluble amyloid oligomers implies common mechanism ofpathogenesis. Science 2003;300:486–9.

ayed R, Sokolov Y, Edmonds B, McIntire R, Milton S, Hall J, et al. Perme-abilization of lipid bilayers is a common conformation-dependent activityof soluble amyloid oligomers in protein folding diseases. J Biol Chem2004:46363.

lunk WE, Jacob RF, Mason RP. Quantifying amyloid �-peptide (A�) aggre-gation using the Congo Red-A� (CR-A�) spectrophotometric assay. AnalBiochem 1999;266:66–76.

oopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, PalsST, van Oers MHJ. Annexin V for flow cytometric detection of phos-phatidylserine expression on B cells undergoing apoptosis. Blood 1994;84:1415–20.

oppel, R., 2003. Alzheimer’s Disease: The Costs to U.S. Businesses in 2002.National Institutes of Health publication number 03-5333. Washington, DC:Alzheimer’s Association, p. 2.

oo DT, Copani AC, Pike CJ, Whitemore ER, Walencewicz AJ, Cotman. Apop-tosis is induced by �-amyloid in cultured central nervous system neurons.Proc Natl Acad Sci 1993;90:7951–5.

artin D, Salinas M, Lopez-Valdaliso R, Serrano E, Recuero M, Cuadrado A.Effect of the Alzheimer amyloid fragment A�(25–35) on Akt/PKB kinaseand survival of PC12 cells. J Neurochem 2001;78:1000–8.

cLaurin J, Franklin T, Fraser PE, Chakrabartty A. Structural transitions asso-ciated with the interaction of Alzheimer’s �-amyloid peptides with ganglio-sides. J Biol Chem 1998;273:4506–15.

ichols HR, Moss MA, Reed DK, Hoh JH, Rosenberry TL. Rapid assemblyof amyloid-� peptide at liquid/liquid interface produces unstable �-sheetfibres. Biochemistry 2005;44:165–73.

’Donovan CN, Tobin D, Cotter TG. Prion protein fragment PrP-(106–126)induced apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells. J Biol Chem 2001;276:23516–23.

allitto M, Murphy RM. A mathematical model of the kinetics of beta amyloidfibril growth from the denatured state. Biophys J 2001;81:1805–22.

hilpott NJ, Turner AJC, Scopes J, Westby M, Marsh JCW, Gordan-Smith EC,et al. The use of 7-amino actinomycin D in identifying apoptosis: simplicityof use and broad spectrum of application compared with other techniques.Blood 1996;87:2244–51.

aynal P, Pollard HB. Annexins: the problem of assessing the biological rolefor a gene family of multifunctional calcium- and phospholipids-bindingproteins. Biochem Biophys 1994;1197:63–93.

oss CA. Protein aggregation and neurodegenerative disease. Nat Med2004;10:S10–7.

ymer DL, Good TA. The role of G protein activation in the toxicity of amy-loidogenic A�-(1–40), A�-(25–35), and bovine calcitonin. J Biol Chem2001;276:2523–30.

chladitz C, Vieira EP, Hermel H, Mohwald H. Amyloid-�-sheet formation atthe air–water interface. Biophys J 1999;77:3305–10.

chmitz T, Friedrich P. Lidocaine induced cell death in a human model of neu-ronal apoptosis. Eur J Anaesthesiol 2002;19:564–70.

elkoe DJ. Alzheimer’s disease: a central role for amyloid. J Neuropathol ExpNeurol 1994;53:438–47.

elkoe DJ. Translating cell biology into therapeutic advances in Alzheimer’s

disease. Nat Alerts 1999;399:A23–31.

ennvik K, Benekikz E, Fastborn J, Sundstrom E, Winblad B, AnkarcronaM. Calcium ionophore A23187 specifically decrease the secretion of �-secretase cleaved amyloid precursor protein during apoptosis in primary ratcortical cultures. J Neurosci Res 2001;63:429–37.

1 urosci

S

SS

T

T

T

T

V

W

W

W

W

W

W

W

W

W

W

0 D. Patel, T. Good / Journal of Ne

hearman MS, Ragan CI, Iversen LL. Inhibition of PC 12 cell redox activityis a specific, early indication of the mechanism of �-amyloid-mediated celldeath. Proc Natl Acad Sci USA 1994;91:1470–4.

hutte B, Nuydens R, Geerts H, Ramaekers F. J Neurosci Meth 1998;86:63–9.immons LK, May PC, Tomaselli KJ, Rydel RE, Fuson KS, Brigham EF, et

al. Secondary structure of amyloid beta peptide correlates with neurotoxicactivity in vitro. Mol Pharmacol 1994;45:373–9.

ang M, Zhang JT. Salvionoic acid B inhibits fibril formation and neurotoxicityof amyloid beta-protein in-vitro. Acta Pharmacol 2001;22:380–3.

ong L, Balazs R, Thornton PL, Cotman CW. �-Amyloid peptide at sublethalconcentrations downregulates brain-derived neurotrophic factor functions incultured cortical neurons. J Neurosci 2004;24:6799–809.

ownsend M, Shankar GM, Mehta T, Walsh DM, Selkoe DJ. Effects of secretedoligomers of amyloid �-protein on hippocampal synaptic plasticity: a potentrole for trimers. J Physiol 2006;572:477–92.

riyoso DH, Good TA. Pulsatile shear stress leads to DNA fragmentation inhuman SH-SY5Y neuroblastoma cell line. J Physiol 1999;515:355–65.

aradarajan S, Yatin S, Aksenova M, Butterfield DA. Alzheimer’s amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity. J StructBiol 2000;130:184–208.

alsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid

beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J BiolChem 1997;272:22364–72.

alsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A,et al. Amyloid �-protein fibrillogenesis: structure and biological activity ofprotofibrillar intermediates. J Biol Chem 1999;274:25945–52.

Z

ence Methods 161 (2007) 1–10

alsh D, Klyubin I, Fadeeva J, Rowan M, Selkoe D. Amyloid beta oligomers:their production, toxicity, and therapeutic inhibition. Biochem Soc Trans2002;30:552–7.

ang S, Becerra-Arteaga A, Good T. Development of a novel diffusion-basedmethod to estimate the size of the aggregated A� species responsible forneurotoxicity. Biotechnol Bioeng 2002a;80:50–9.

ang H, Zhang Y, Cao L, Han H, Wang J, Yang B, et al. HERG K+ chan-nel, a regulator of tumor cell apoptosis and proliferation. Cancer Res2002b;62:4843–8.

ang SS, Kazantzi V, Good TA. A kinetic analysis of the mechanism of �-amyloid induced G protein activation. J Theor Biol 2003;221:269–78.

ard RV, Jennings KH, Jepras R, Neville W, Owen DE, Hawkins J, et al. Frac-tionation and characterization of oligomeric, protofibrillar and fibrillar formsof �-amyloid peptide. Biochem J 2000;348:137–44.

eiss JH, Pike CJ, Cotman CW. Ca2+ channel blockers attenuate beta-amyloidpeptide toxicity to cortical neurons in culture. J Neurochem 1994;62:372–5.

hite AR, Zheng H, Galatis D, Maher F, Hesse L, Multhaup G, et al. Sur-vival of cultured neurons from amyloid precursor-protein knock-out miceagainst Alzheimer’s �-amyloid toxicity and oxidative stress. J Neurosci1998;18:6207–17.

illiams AD, Sega M, Chen M, Kheterpal I, Geva M, Berthelier V, Kaleta DT,

Cook KD, Wetzel R. Structural properties of Abeta protofibrils stabilized bya small molecule. PNAS 2005;102:7115–20.

hang HY, Brimijoin S, Tang XC. Apoptosis induced by �-amyloid 25–35 inacetylcholinesterase-overexpressing neuroblastoma cells. Acta PharmacolSin 2003;24:853–8.