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Role of aggregation conditions in structure, stability, and toxicity of intermediates in the A fibril formation pathway

¹ Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843-3122, USA
² Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, USA
³ Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, Maryland 21250, USA

(RECEIVED August 23, 2006; FINAL REVISION December 8, 2006; ACCEPTED December 19, 2006)

	Abstract

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

 
-amyloid peptide (A) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). A readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of A aggregates or oligomers is still under investigation. In this article, we show that different A incubation conditions in vitro can affect the rate of A fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, A aggregates faster than A prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during A aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or A fibrils formed with agitation. In addition, A fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the A aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between A aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic A oligomers.

Keywords: Alzheimer's disease; amyloid; aggregation; guanidine hydrochloride; unfolding

	Introduction

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

 
Alzheimer's disease (AD) is a progressive, neurodegenerative disease of the central nervous system and the leading cause of dementia in aging population. One of the histopathological hallmarks of AD is the formation of neuritic plaques, the major protein component of which is -amyloid peptide (A). Two variants of A, A1-40 and A1-42, are the most abundant forms found in AD (Bayer et al. 2001; Turner et al. 2003).

Accumulating data show that while A aggregates readily into amyloid fibrils, the A species in soluble oligomeric forms rather than fibrils are closely associated with the neurotoxicity in vivo and in vitro. Toxicity has been attributed to A-derived diffusible ligands (ADDLs) composed of A1-42, with molecular weights between 17 and 42 kDa (Lambert et al. 1998; Klein et al. 2001) and heterogeneous globular species (also described as ADDLs) with hydrodynamic radii between 3 and 8 nm (Chromy et al. 2003). Spherical aggregates, not described as ADDLs, with radii over 15 nm have also been reported to be toxic (Hoshi et al. 2003). Toxic protofibrils of A1-40 have been described with hydrodynamic radii ranging from 9 nm to over 300 nm (Walsh et al. 1999; Ward et al. 2000; Wang et al. 2002). When investigators have compared toxicity of fibril and oligomer A species, they have found that the oligomeric species were more toxic (Dahlgren et al. 2002). While some investigators believe that there is a common structure associated with amyloid toxicity (Kayed et al. 2003), a careful characterization of such a common structure is still not available. Part of the challenge in elucidating the relationship between A structure and toxicity is the difficulty in isolating pure samples of A oligomers for characterization, although use of urea (Kim et al. 2004), low temperatures (Yong et al. 2002), or photocross-linkers have aided in their identification (Bitan and Teplow 2004). One approach has been to carefully characterize A monomers and fibrils, and from those structures infer the nature of a toxic aggregation intermediate. However, it is becoming increasingly clear that even the molecular level structure of A fibrils changes with aggregation conditions (Stine et al. 2003; Petkova et al. 2005, 2006; Shivaprasad and Wetzel 2006; among others).

In work presented here, we examined the differences in A aggregation intermediates and final structures formed when only a simple modification in A aggregation conditions was made, the presence or absence of agitation during aggregation. We show that while the final structures in the A aggregation pathway are comparable by measures such as electron microscopy, the toxicity of fibrils formed are significantly different. In addition, intermediates in the aggregation pathway show significantly different structural rearrangements. When guanidine hydrochloride unfolding was used as a simple measure of stability of different aggregated species, the A aggregation intermediates formed under quiescent conditions, the most toxic A species we observed, were the structures that changed conformation at the lowest concentrations of guanidine hydrochloride. These results highlight the differences in A aggregation mechanisms when aggregation occurs with agitation or under quiescent conditions. In addition, our results provide insight into the structure of the toxic A oligomers formed during aggregation under quiescent conditions.

	Results

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

 
Kinetics of A aggregation with and without mixing
We examined the structure of 100 µM A1-40 samples when aggregated with and without agitation at 37°C. Congo red binding was used as an indicator of extended -sheet structure and changes in A aggregation with time. As seen in Figure 1, the kinetics of A fibril formation, as indicated by Congo red binding, follow a similar trend with and without agitation. However, the time scales for fibril formation under the two conditions differed by a factor of 2 to 4. In both cases, fibril formation followed a characteristic sigmoidal curve, with a lag phase at early incubation times, followed by a fibril growth phase, then a saturation phase. The lag phases were about 4 h and 16 h for agitation and quiescent aggregation conditions, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 1. A aggregation as measured by Congo red binding as a function of time after initial dissolution of the peptide. One hundred millimolar A was incubated at 37°C (A) with gentle agitation and (B) under quiescent conditions.

Size characterization of A species during aggregation

View larger version (34K): [in this window] [in a new window]   Figure 2. Representative size exclusion chromatograms of 100 µM A at different times during aggregation. A samples were incubated at 37°C for (A) 2, 4, 8, and 24 h with gentle agitation and (B) 4, 8, 24, and 72 h under quiescent conditions.

View this table: [in this window] [in a new window]   Table 1. Percentage of A species in peptide samples aggregated for different lengths of time as determined from peak areas of SEC chromatograms

Electron micrographic images of A species during aggregation

View larger version (95K): [in this window] [in a new window]   Figure 3. Representative EM images of 100 µM A at different times during aggregation at 37°C; (A) fresh A, (B) 2 h of aggregation with gentle agitation, (C) 24 h of aggregation with gentle agitation, (D) 8 h of aggregation under quiescent conditions, and (E) 72 h of aggregation under quiescent conditions.

Changes in A secondary structure during aggregation

View larger version (31K): [in this window] [in a new window]   Figure 4. Secondary structure of A at different times during aggregation with gentle agitation (A) and under quiescent conditions (B). All times indicate time of incubation at 37°C after initial dissolution of 100 µM A. Representative CD spectra are shown. (Light thin solid line) fresh A; (dark thin solid line) 2 h; (light thick solid line) 4 h; (dark thick solid line) 8 h; (light thick broken line) 10 h; (dark thick broken line) 12 h.

Stability of A species to structure change in denaturant

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of guanidine hydrochloride (GuHCl) on structure of different A aggregates as measured by CD intensity at 220 nm. A samples (100 µM) were aggregated either with gentle agitation or under quiescent conditions at 37°C for various lengths of time, after which A samples were mixed with GuHCl to a final concentration of 10 µM A and 0.5–7 M GuHCl. CD intensity at 220 nm of A samples in GuHCl were monitored for 2 h, until no further detectable changes in structure or CD intensity occurred. CD intensities at 220 nm were used for A stability tests. (Filled triangles) fresh A; (open squares) A aggregated with gentle agitation for 24 h; (filled circles) A aggregated for 8 h under quiescent conditions.

Biological activities of A species

Relative cell viabilities of cells treated with A samples aggregated under different conditions are shown in Figure 6. Cells treated with fresh A had almost the same cell viability (102% ± 9%) as negative control population (p > 0.05). A species prepared at different times of aggregation with agitation were significantly more toxic than controls (p < 0.05), and toxicity increased with time of aggregation of the peptide (Fig. 6A). When A was aggregated under quiescent conditions, a different pattern of toxicity with time of aggregation was observed (Fig. 6B). The toxicity of species formed within the first 8 h of aggregation under quiescent conditions increased to a maximum observed toxicity at 8 h. Species formed after 24 or more hours of aggregation, however, had significantly less toxicity than those formed at earlier times. In addition, the toxicity of fibrils formed with agitation (24 h) was significantly greater than the toxicity of fibrils formed under quiescent conditions (72 h) (p < 0.05).

View larger version (14K): [in this window] [in a new window]   Figure 6. Relative cell viability of differentiated SY5Y cells treated with 100 µM A that had been aggregated with gentle agitation (A) or under quiescent conditions (B) for different lengths of time prior to addition to cells. Cells were incubated with A for 2 h prior to assessment of viability via staining with annexin and 7AAD followed by flow cytometry.

	Discussion

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

As seen in Figure 1, kinetics of A aggregation, regardless of the presence or absence of agitation, differ in scale, but appear similar in form. The characteristic lag in development of extended -sheet structure to which Congo red binds is typical of a nucleation mechanism of aggregation that a number of investigators propose for A fibril formation (Lomakin et al. 1997; Pallitto and Murphy 2001; Pellarin and Caflisch 2006). Increased rates of aggregation with agitation relative to A aggregation under quiescent conditions has also been observed by others (Tycko 2006; Petkova et al. 2006). In this study, we provide agitation by rotation at 18 rpm on a laboratory rotator. Others have used gentle agitation and sonication to mix during A aggregation and found that the higher energy mixing increased the rate of aggregation (Petkova et al. 2006). While Congo red binding suggests that aggregation with and without agitation may be similar, data obtained from size exclusion chromatography, CD spectroscopy, and electron microscopy (Figs. 2–4) suggest that there are significant differences in the mechanism of aggregation with and without agitation of A.

SEC chromatograms indicate that, throughout aggregation under conditions with or without agitation, species that elute at volumes consistent with monomer and dimer are present in the same ratio of peak areas in all chromatograms. This indicates these two species were in equilibrium with each other under all these conditions. Others have made similar observations of A aggregation (Pallitto and Murphy 2001). The amounts of monomer and dimer relative to other species decreased with time, corresponding to increases in fibril formation as indicated by Congo red binding (Table 1). Different investigators have suggested that monomer or dimer are the building blocks for fibril nucleation or growth (Garzon-Rodriguez et al. 1997; Tseng et al. 1999; Roher et al. 2000; Hwang et al. 2004).

CD data indicate that A was largely -sheet prior to aggregation and did not undergo significant secondary structural rearrangements when aggregated with agitation. When aggregated under quiescent conditions, however, a dramatic shift in structure was observed at intermediate times (4–8 h after aggregation is initiated), which corresponded to the presence of an intermediate-sized species in chromatograms (approximate molecular weight as estimated from elution volume on SEC of 53 kDa) and large globular species in electron micrographs. None of these intermediate species were observed when aggregation proceeded with agitation.

A number of investigators have observed A aggregation intermediates, formed either at low temperature or via a particular preparation method, sometimes referred to as A derived diffusible ligands (ADDLs) (Lambert et al. 1998; Klein et al. 2001), that have molecular weights within the range we observed via SEC. A number of reports indicate that these ADDLs are the (or one of the) toxic A oligomers (Lambert et al. 1998). Others have described spherical species of various sizes that are A aggregation intermediates with sizes ranging from 3 to 20 nm, and some up to 100 nm depending on starting peptide and preparation method (Harper et al. 1999; Westlind-Danielsson and Arnerup 2001; Hoshi et al. 2003). Toxicity of the spherical A species has also been reported (Hoshi et al. 2003). The 53-kDa species observed via SEC may be distinct from the spherical species observed via electron microscopy; however, it is not possible to tell from our data. Others have observed the formation of A micelles (Lomakin et al. 1996; Yong et al. 2002; Kim and Lee 2004; Kim et al. 2004; Sabate and Estelrich 2005) which might appear as a large globular species via EM, but could change apparent molecular size during chromatography. Alternately, it is probable that via SEC we would not be able to detect a globular protein aggregate of 20 nm diameter as it would be removed from our sample during our sample pretreatment, nor would we be able to detect 53-kDa proteins via EM as they would be below the resolution of the microscope used.

That we observe dramatic structural rearrangements with aggregation under quiescent conditions but did not observe the same structural rearrangements when aggregation occurred with agitation is not surprising. If A aggregation is nucleation dependent as others propose (Lomakin et al. 1997; Ramírez-Alvarado et al. 2000; Wogulis et al. 2005), then with agitation, one would expect more rapid collisions of molecules and faster nucleation, both in solution and against the walls of the tube in which aggregation took place. The faster rate of nucleation would mean that A would have less time to undergo any intramolecular structural rearrangements that might be energetically favorable and increase stability of the fibril formed. Without agitation, the rate of peptide collision with other peptides would be slower, allowing more time for intramolecular rearrangement of the peptide before combining with other peptides to form an aggregation intermediate. In short, agitation would favor intermolecular rearrangements to bury hydrophobic surfaces or form hydrogen bonds that would lead to aggregation, while quiescent conditions would favor intramolecular rearrangements to bury hydrophobic surfaces. These intramolecular rearrangements could lead to formation of aggregation intermediates not seen when intermolecular interactions predominate.

It has recently been suggested that amyloid fibrils form through a micelle intermediate when relatively unstable -sheet-forming peptides form the building blocks of the amyloid, while no micelle intermediate would be seen if a stable -sheet-forming peptide were used as fibril precursors (Pellarin and Caflisch 2006). It is possible that under quiescent conditions, A forms a somewhat unstable monomer, but with agitation, a more stable -sheet monomer or dimer is formed.

Work coming out of the laboratories of Tycko (Petkova et al. 2005, 2006) and Wetzel (Whittemore et al. 2005; Shivaprasad and Wetzel 2006; Williams et al. 2006) demonstrates that A fibrils formed with agitation and those formed under quiescent conditions have different molecular level structures. The presence of intramolecular salt bridges, the number of layers of -sheets that form a protofibril unit, and peptide residues that make up -sheet segments may all be different in fibrils formed under the different conditions. Thus it is not surprising that our data suggest that different mechanisms of aggregation govern fibril formation with and without agitation.

The relative stability of species formed during aggregation in the presence and absence of agitation can be inferred from data collected on conformation change of A in a denaturant (Fig. 5; Table 2). When A was aggregated with agitation, fibrils underwent conformation change in denaturant more readily than monomer. The trend in change in protein conformation with denaturant concentration (or slope of changes in free energy with denaturant concentration "m") is traditionally interpreted as a measure of buried surface (Myers et al. 1995; Pace et al. 1996), suggesting that A fibrils formed during agitation have more buried surface than A monomers. When A was aggregated under quiescent conditions, aggregation intermediates underwent conformation change more easily than either fresh or fibril A, suggesting the intermediate had the most buried surface. While this interpretation of our results is in disagreement with an earlier report (Kremer et al. 2000), it is consistent with data that comes from our laboratory of the fluorescence of the hydrophobic probe, 1-anilino-8-naphthalene sulfonic acid (ANS), indicating that the aggregation intermediate formed under quiescent conditions was less hydrophobic or bound less ANS than either monomer or fibril (unpubl. results).

We examined biological activity or toxicity of fibrils and aggregation intermediates formed with and without agitation (Fig. 6). Toxicity of fibrils was greater when formed with agitation, while toxicity of aggregation intermediates formed between 4 and 8 h after initiation of aggregation under quiescent conditions were more toxic than other species formed. Fibrils formed with agitation and aggregation intermediates formed under quiescent conditions changed structure more readily in denaturant than other species examined (Fig. 5). Thus, for samples and conditions considered here, the difference in apparent stability in denaturant correlated with toxicity. A number of investigators have suggested that A membrane interactions via A conformational changes are important in the mechanism of A toxicity (McLaurin and Chakrabartty 1997; Terzi et al. 1997; Demuro et al. 2005). Behavior of A in a membrane and in a denaturant might be analogous. Thus, one could speculate that toxicity of an A species may be related to its ability to change structure within the cell membrane.

There are several alternative explanations of the data presented. Others have reported that A oligomers with structures similar to those we describe forming during aggregation under quiescent conditions are more toxic than fibrils (Dahlgren et al. 2002; Hoshi et al. 2003). However, some investigators have observed that fibrils formed under quiescent conditions, then sonicated, are more toxic than fibrils formed with agitation (Petkova et al. 2005). When comparing toxicity of fibrils formed via different methods, it has been very difficult to characterize concentration or number of fibrils in a solution. The average length of fibrils has been shown to change with aggregation condition (Walsh et al. 1999; Ward et al. 2000). The number of fibrils might also change with aggregation conditions (or with sonication). With agitation, one would expect larger numbers of fibrils or fibril nuclei than would be formed without agitation. Larger number of fibril ends or fibril nuclei would be expected to be associated with greater toxicity (Wogulis et al. 2005). Consequently, differences in toxicity observed may simply be due to differences in oligomer or fibril concentrations in solution.

In summary, we report structure and toxicity of A species formed during aggregation via different methods. We show that fibrils formed via different methods do not have the same toxicity nor the same apparent stability to denaturants, nor do they appear to be formed via the same mechanism or have the same intermediate aggregation species. Aggregation intermediates formed under quiescent conditions appear to be similar to A oligomer species reported by others such as ADDLs or spherical globules. They also may be the micelles suggested by others to be part of the A aggregation pathway from which nuclei form. The same aggregation intermediates had low -sheet content, low stability in denaturants, and high toxicity. This work provides insight into the structure of toxic A oligomers and highlights differences in aggregation mechanisms of A fibrils formed with and without agitation.

	Materials and Methods

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

 
Materials
-Amyloid (A)–(1-40) was purchased from AnaSpec. Human neuroblastoma SH-SY5Y cells were purchased from ATCC (CRL-2266). Cell culture reagents were purchased from Invitrogen Life Technologies. Superose6 HR 10/30 Column was purchased from Amersham Pharmacia Biotech. All other disposable supplies for FPLC were purchased from Amersham Pharmacia Biotech. Congo red was purchased from Fisher Chemicals. All other chemicals, unless otherwise specified, were obtained from Sigma-Aldrich Co.

Protein sample preparation
Lyophilized A1-40 was freshly dissolved in 0.1% (v/v) Trifluoroacetic acid (TFA) solvent at the concentration of 10 mg/mL. This solution was incubated at room temperature for 20–30 min in order to completely dissolve the A. Filtered phosphate-buffered saline (PBS, 4.3 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl, 1.4 mM KH₂PO₄ at pH 7.4) was added to the A solution to make the final concentrations used in experiments. For cell viability assays, MEM medium was used instead of PBS buffer. For aggregation with gentle mixing, A samples were mixed in 1-mL vials filled 0.6 mL of solution mounted on a rotator at 18 rpm and 37°C, and samples were taken out with time. For aggregation under quiescent conditions, A samples were incubated without disturbance in a 37°C incubator.

Congo red binding
Congo red was dissolved in PBS at the concentration of 120 µM and syringe filtered. The Congo red solution was mixed with protein samples at 1:9 ratios to make the final concentration of Congo red 12 µM. After a short vortex, the mixtures were incubated at room temperature for 30–40 min. Absorbance was measured from 400 nm to 700 nm (UV-Vis spectrometer model UV2101, Shimadzu Corp.). Alternatively, Congo red absorbance was read at 405 nm and 540 nm using an Emax Microplate Reader (Molecular Devices). In both cases, PBS buffer was used as blank control. The concentration of A fibrils was estimated from Congo red binding via Equation 1:

where ⁵⁴¹A_t and ⁴⁰³A_t are the absorbances of the Congo red-A mixtures at 541 nm and 403 nm, respectively, and ⁴⁰³A_CR is the absorbance of Congo red alone in phosphate buffer (Klunk et al. 1999). When using the microplate reader, absorbances at 405 nm and 540 nm were assumed to be same as those at 403 nm and 541 nm.

Size exclusion chromatography (SEC)
A Superose6 HR 10/30 Column in a FPLC System (Pharmacia) was used to determine the molecular size of A species. PBS buffer was used as a mobile phase with a flow rate of 0.5 mL/min. A 100-µL sample loop was used. A samples were centrifuged at 7000g for 30 sec prior to SEC analysis. Supernatants of centrifuged samples were loaded in the 100-µL loop and injected into the column. A species were detected by UV detector at 254 nm. The following proteins were used for calibration standards: ribonuclease (13.7 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), and albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (660 kDa), and BD 2000 (2000 kDa). To calculate relative areas of A in peaks from the SEC chromatograms, first the total area of a 100-µM A sample was obtained from a chromatogram obtained without a column in the FPLC system. On the basis of this total area, relative areas from the SEC chromatograms were calculated.

Circular dichroism (CD)
Secondary structure of A was measured using a PiStar-180 Circular Dichroism Spectrometer (Applied Photophysics). CD spectra in the far UV range (190–260 nm) were obtained using a 1-cm quartz cell, at 37°C, using a Xe lamp as a light source, a 1.0-nm bandwidth, 1.0-nm step interval, and 1.5 sec/nm scanning speed. The spectrometer was purged with nitrogen gas during measurement. PBS buffer was used for the calibration.

Electron micrograph (EM)
Two hundred microliters of A peptide solution, prepared as described above, were mixed, placed on glow discharged grids, and then negatively stained with 1% aqueous ammonium molybdate (pH 7.0). Grids were examined in a Zeiss 10C transmission electron microscope at an accelerating voltage of 80 kV. Calibration of magnification was done with a 2160 lines/mm crossed line grating replica (Electron Microscopy Sciences).

A stability with guanidine hydrochloride (GuHCl)
One hundred micromolar A samples were prepared as described in the protein sample preparation. A samples at 37°C were measured at 0 (fresh A) and 24 h after initial dissolution for agitated samples and 4, 8, and 72 h after dissolution for samples aggregated under quiescent conditions. GuHCl was dissolved in PBS buffer and prepared at different concentrations. A samples were mixed with GuHCl solution at the ratio of 1:9. The final A concentration in each sample was 10 µM. The final GuHCl concentrations varied from 0.5 M to 7 M. A samples in different GuHCl concentrations were incubated at 37°C for 2 h with the GuHCl. CD spectra were taken of the incubated A-GuHCl samples and each was calibrated against a solutions of GuHCl at the same concentration in PBS buffer. CD intensity at 220 nm, a typical minimum for -sheet structures, was used as an indicator of change in protein structure with denaturant addition. The midpoint of the denaturation curve, GuHCl_1/2, was taken as the concentration of denaturant at which half of the total change in CD absorbance upon unfolding was observed. To determine GuHCl_1/2, the linear portion of the denaturant curve was fit to a straight line, the slope of which was used to estimate the concentration of GuHCl at which one half of the total change in CD absorbance was observed.

Cell culture
SH-SY5Y cells were grown in Minimum Essential Medium (MEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 25 mM sodium bicarbonate, 100 units/mL penicillin, and 100 mg/mL streptomycin. Cells were cultured in a 5% (v/v) CO₂ environment at 37°C incubator. Low passage number cells were used (<p20) in all experiments to reduce instability of the cell line.

Biological activity assay
For biological activity tests, SH-SY5Y cells at a density of 1 x 10⁵cells/well were grown in 96-well plates. Cells were fully differentiated by addition of 20 ng/mL NGF for 8 d. A samples in MEM medium were added to the differentiated SY5Y cells and the cells were incubated with A samples at 37°C for 2 h. Negative controls (cells in medium with no A) and positive controls (cells treated with 800 µM H₂O₂ in 50% [v/v] medium for 2 h) were also prepared. At least 3 wells were prepared for each A treatment and each positive and negative control.

Cell viability was determined by using two fluorescent dyes, Annexin V-PE and 7-Amino-actinomycin (7-AAD). Annexin V-PE is a Ca²⁺ dependent phospholipid binding protein that has high binding affinity for phosphatidylserine on apoptotic cells. 7-AAD is taken up by necrotic or damaged cells, whereas live cells exclude 7-ADD. In this experiment, cells unstained for both Annexin V-PE and 7-AAD were taken as live cells. In order to stain the dead cells, A treated cells were washed with PBS 1 or 2 times and 1x binding buffer was added to the cells (1x binding buffer: 10 mM HEPES/NaOH at pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). Five microliters of Annexin V-PE and 5 µL of 7-AAD were added to the cells and cells were incubated for 15–20 min at room temperature in the dark. One hundred fifty microliters of 2x binding buffer were added to the cells, and cells were collected using a scrapper. Stained cells in 96-well plates were loaded in the FACS array (BD FACSArray, BD Bioscience) and fluorescence histograms for cells were obtained. To set up compensation and gates for the cell viability assays, three sets of staining controls were prepared, unstained cells, cells stained with Annexin V-PE alone, and cells stained with 7-AAD alone. Cells not stained with either dye (population with low fluorescence intensities for Annexin V-PE and 7-AAD) were taken as live cells, and the relative cell viabilities were calculated using Equation 2:

where L.C._sample is live cells (%) of A treatment, L.C._Ncontrol is live cells (%) of negative control, and L.C._H2O2 is live cells (%) of H₂O₂ treatment (positive control).

Statistical analysis
For each experiment, at least three independent determinations were made. Significance of results was determined via the t test with p < 0.05 (95% confidence interval) unless otherwise indicated. Data are plotted as the mean plus or minus the standard error of the measurement.

	Footnotes

 
⁴ Present address: Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.

Reprint requests to: Theresa Good, Chemical and Biochemical Engineering, UMBC, 1000 Hilltop Circle, Baltimore, MD 21250, USA; e-mail: tgood{at}umbc.edu; fax: (410) 455-1049.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062514807.

	Acknowledgments

TOP Abstract Introduction Results Discussion Materials and Methods Acknowledgments References

 
This work was supported by a grant from the National Institutes of Health (R01 NS042686) to T.A.G. and E.J.F. We thank Dhara Patel at UMBC for her assistance in flow cytometry analysis and Ann Ellis of the microscopy imaging center at Texas A&M University for preparation of electron micrograph images.

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