Screening Compounds for Apoptotic and Cytotoxic Activity Using the Guava PCA-96, a Novel Benchtop Personal Cell Analysis System

Dianne M. Fishwild, Guava Technologies, Inc.

Abstract

A panel of 80 anti-inflammatory, anti-neoplastic and anti-infective compounds were screened for their ability to cause cell death or apoptosis induction using rapid and objective 96-well plate assays that measure cell concentration and viability (Guava® ViaCount®) or Annexin V binding to apoptotic and dead cells (Guava Nexin®).  The Guava PCA™-96, a novel benchtop microcapillary cell analysis system, was used to acquire the data for both assays.  Compounds were characterized for potency (IC50 or EC50), efficacy (percent killed or percent positive for apoptosis), and specificity (which cells were affected). Twenty-three drugs exhibited minimal to substantial activity on both cell lines inducing either apoptosis, cytotoxicity or both; four were effective against only CHO cells and ten against only Jurkats.  Moreover, Guava ViaCount and Guava Nexin, both of which detect apoptosis induction and cytotoxicity, albeit in different ways, could be used in combination to confirm the specific activity of the drugs or other compounds as well as to identify potential false positives.

These results illustrate that investigation of cytoactive compounds in cell-based assays can be done quickly and effectively using the automated, microplate-based benchtop cell analysis system, the Guava PCA-96 in conjunction with the mix and read Guava ViaCount and Guava Nexin assays.

Introduction

Bottlenecks in drug discovery and drug development occur at key points where detailed information about cellular behavior is required.  Many available cell-based assays report on analyses of “bulk” populations, but very few options enable automated analysis of single cells, particularly live cells or cells in suspension.  The Guava PCA-96, an automated cell analysis platform for 96-well plates, enables users to apply the principles of capillary flow analysis to microplate-based assays.

The Guava PCA-96 system performs a number of automated cellular assays, including viability, absolute cell counting, three apoptotic assays and a cell cycle assay. Two of these assays, the Guava ViaCount and Guava Nexin assays, were tested here.  The Guava ViaCount assay rapidly and reliably determines cell concentration and viability. The Guava ViaCount Flex reagent used for this assay consists of two DNA-binding fluorescent dyes that differentially stain viable and non-viable (dead or apoptotic) cells based on cell membrane permeability.  The Guava PCA-96 system counts nuclei stained with the membrane permeant DNA dye, and then distinguishes cells from bare nuclei, or DNA-associated cell debris using forward scatter (FSc) signals. That combination allows Guava ViaCount to accurately determine cell concentration.  The system can determine the viability of the cell sample since it can distinguish non-viable cells that will also absorb the second dye. Some apoptotic cells can also be distinguished in the Guava ViaCount assay because they show intermediate uptake levels of the membrane impermeant dye, absorbing more dye than the viable cells, but less than dead cells.  The Guava ViaCount assay more accurately and precisely determines cell concentrations and viabilities than Trypan Blue exclusion1, especially at low cell concentrations, and shows much less operatorto-operator variability, in part because Trypan Blue has been reported to overestimate cell viability2,3.  Thus, the Guava ViaCount assay is sufficiently robust and precise for screening cytotoxic and cytoactive compounds.

Apoptosis, or programmed cell death, is an important and active regulatory pathway of cell growth and proliferation. Cells respond to specific induction signals by initiating intracellular processes that result in characteristic physiological changes. Among these are externalization of phosphatidylserine (PS) to the cell surface, cleavage and degradation of specific cellular proteins, compaction and fragmentation of nuclear chromatin, and loss of membrane integrity (in late stages).4-8

Early in the apoptotic pathway, molecules of PS are translocated to the outer surface of the cell membrane. The Guava Nexin Assay monitors this externalization of PS in addition to the loss of membrane integrity that occurs later in the process.  The Guava Nexin reagents consist of 1) Annexin V, a calcium-dependent phospholipid binding protein with a high affinity for PS,9-11 conjugated to phycoerythrin (PE) and 2) 7-amino actinomycin D (7-AAD) which can bind to intracellular nucleic acid once the membrane integrity has been breached.  The Guava Nexin assay utilizes Annexin V-PE to detect PS on the external membrane of apoptotic cells12-17. The 7-AAD is excluded from live, healthy cells and early apoptotic cells, but permeates late stage apoptotic and dead cells.18 Three populations of cells can be distinguished in this assay:

  • Non-apoptotic cells: Annexin V (–) and 7-AAD (–)
  • Early apoptotic cells: Annexin V (+) and 7-AAD (–)
  • Late stage apoptotic and dead cells: Annexin V (+) and 7-AAD (+)

The Guava Nexin assay can thus be used to screen compounds for both early and late apoptotic activity.

We tested a panel of 80 small molecule known drugs in a combinatory screen designed to identify “hit” compounds for cytotoxicity or apoptosis induction using both the Guava Nexin and Guava ViaCount assays.  We compared the activity of these compounds at two time points against two different cell lines and examined whether cytotoxic compounds were also apoptotic inducers and vice versa.  We subsequently titrated some of the “hit” compounds to determine their potency. 

Methods and Materials

Cells, Materials and Compounds

A non-adherent human T cell line, Jurkat (ATCC Cat. No. TIB-152), and an adherent hamster fibroblast line, CHO-K1 (ATCC Cat. No. CCL-61), were used for these studies.  Cells were kept in log phase growth in medium designed to stimulate optimal growth.  RPMI medium (Cellgro Cat. No. MT 10-04-CV) supplemented with 10% FBS (Cellgro Cat. No. MT 35-010-CV), 2 mM L-glutamine (Cellgro Cat. No. MT 25-005-CV), 4.5 g/L glucose (Sigma Cat. No. G8769) and 1 mM sodium pyruvate (Cellgro Cat. No. MT 25-000-CL) was used for culturing Jurkats and F12K medium (Cellgro Cat. No. MT 10-025-CV) supplemented with 10% FBS and 2 mM L-glutamine was used for culturing CHO cells.  For the Guava ViaCount and Guava Nexin assays, Jurkat cells were cultured in 96-well flat bottom polystyrene plates (Falcon Cat. No. 352072) and CHO cells were cultured in 96-well flat bottom polystyrene plates coated with hydrogel to prevent their attachment (Corning Costar Cat. No. 3474).  A known inducer of apoptosis, staurosporine (Calbiochem Cat. No. 569397), was used as a positive control.  A panel of 80 cytotoxic, immunosuppressive, antiproliferative and anti-inflammatory compounds was obtained from MicroSource Discovery Systems, Inc.  For initial screening, compounds which were at 10 mM in DMSO were diluted in two steps to 10 µM in cell culture medium just prior to use.   For hit confirmation and potency determination, selected compounds were titrated out to the indicated concentrations.

Assays

Guava ViaCount.  For compound screening experiments, between 100 and 200 cells/µL in 50 µL of appropriate complete medium were added to replicate 96-well plates with the panel of 80 small molecule compounds (in wells A2 through H11), 0.05% sodium azide as a positive control (in wells A12, B12, C12, F1, G1, and H1), diluent controls (0.5% DMSO only, in wells D1, D12, E1 and E12), and medium only (in wells A1, B1, C1, F12, G12 and H12) in 50 µL and incubated at 37 ˚C, 5% CO2 for various times.  Plates were incubated for 4 or 24 hours (Jurkats) or 20 and 41 hours (CHO cells).  At the end of culture, Guava ViaCount Flex (Guava Cat. No. 4500-0110) was added at a final dilution of 1:100 (Jurkat) or 1:50 (CHO) to the cells and mixed.  After a 20 minutes incubation at RT in the dark, the plates were loaded on Guava PCA-96 Systems and 1000 events per well were acquired.

Guava Nexin.  Approximately 50,000 cells/well in appropriate complete medium were added to replicate 96-well plates and incubated at 37 ˚C, 5% CO2 for the indicated times with the panel of 80 small molecule compounds (in wells A2 through H11), 5 µM staurosporine as a positive control (in wells A12, B12, C12, F01, G01 and H01), and diluent (0.5% DMSO only) and medium only as negative controls (in wells D1, D12, E1 and E12 and in wells A1, B1, C1, F12, G12 and H12, respectively).  Plates were incubated for 4 or 24 hours (Jurkats) or 20 and 41 hours (CHO cells).  The Guava Nexin Kit reagents (Guava Cat. No. 4500-1600), Annexin V-PE, 7-AAD and a high Ca2+ containing buffer, were then added to the cells in one solution (200 µL per well of 3 mL Annexin V-PE, 1.5 mL 7-AAD and 36.5 mL 1X Nexin buffer), mixed, and incubated for 25 minutes in the dark.  Two thousand events per well were subsequently acquired on each Guava PCA-96 System.

Results

We used the Guava Nexin assay to screen a panel of small molecules for apoptotic activity.  The panel of 80 cytoactive compounds and additional control compounds were assessed at two time points and for the induction of early apoptosis (Annexin V+, 7-AAD–) and of late apoptosis or death (Annexin V+, 7-AAD+).  Compounds were also assessed at two time points with the Guava ViaCount assay, which readily detects viable and dead cells.  This assay can also detect late stage apoptotic cells whose membranes are beginning to break down and hence allow passage of an intermediate amount of the membrane impermeant dye.

Figure 1 shows examples of the results obtained with Jurkat cells for the negative control and three test compounds assessed using the Guava ViaCount and Guava Nexin assays.  As shown in the Guava ViaCount plot for the negative control, the majority of the cells were viable and appeared to the left of the solid red marker.  These cells had absorbed the nucleated cell dye but not the membrane impermeant dye (the viability dye).  In the Nexin plot, the viable cells appeared in the lower left quadrant because they bound neither the Annexin V-PE nor the 7-AAD dyes. 

Figure 1: Examples of the Effects of “Hit” Compounds on Jurkat Cells. The effects of selected compounds at 24 hours are shown for the Guava ViaCount assay on the left and for the Guava Nexin assay on the right.

Figure 1: Examples of the Effects of “Hit” Compounds on Jurkat Cells. The effects of selected compounds at 24 hours are shown for the Guava ViaCount assay on the left and for the Guava Nexin assay on the right.

5-Azacytidine induced apoptosis and cell death, shifting the pattern of cells displayed.  The ViaCount plot shows that a large number of dead cells stained with the membrane impermeant viability dye and appeared to the right of the purple marker.  As seen in the Nexin plot, the apoptotic cells had bound the Annexin V-PE and appeared in the lower right quadrant. The dead cells, which had also taken up the 7-AAD, appeared in the upper right quadrant.  While the ViaCount plot did not clearly show the apoptotic cells induced by 5-azacytidine, there was a slight shift in the staining pattern of the viable cells, suggestive of an effect.  The Guava ViaCount assay appeared to detect apoptotic cells that have advanced beyond the stage of PS translocation.

For the length of induction in this experiment, mebendazole induced apoptosis without significant cell death, though not as strongly as did 5-azacytidine.  Like 5-azacytidine, mebendazole did not induce a clear apoptotic staining pattern in the Guava ViaCount assay even though it clearly induced apoptosis, based on the translocation of PS.

Ellagic acid showed no apoptotic activity in the Guava Nexin assay, although there was a downward shift in the staining pattern of the cells in the Guava ViaCount assay, consistent with less uptake of the nucleated cell dye.  This pattern of results suggests that ellagic acid was either inducing apoptosis without altering the location of PS, or that ellagic acid was a false positive and is having a non-specific effect on the nucleated dye uptake.  One can thus use the changes in patterns observed on the dot plots to determine the extent to which various compounds induce apoptosis and death.

The results for the 80 compounds and 16 controls screened with the Guava Nexin assay at 4 and 24 hours are summarized in Figure 2.  The percentage of early apoptotic cells in the negative control wells (n=10) was 2.6% at both time points; approximately 1% of the cells were late apoptotic and dead cells. In the positive control wells (n=6), 96% and 35% were in early apoptosis at 4 and 24 hours, respectively; late apoptotic plus dead cells counted for 2% and 64% of the cells at the same time points.

Figure 2: Detection of Apoptosis Induction in a Panel of Active Compounds. Jurkat cells were cultured for either 4 hours (A, C) or 24 hours (B, D) and then analyzed for the presence of cells in early apoptosis (A, B) or late apoptosis/death (C, D).

Figure 2: Detection of Apoptosis Induction in a Panel of Active Compounds. Jurkat cells were cultured for either 4 hours (A, C) or 24 hours (B, D) and then analyzed for the presence of cells in early apoptosis (A, B) or late apoptosis/death (C, D).

The data generated were extremely reproducible as the replicate plates showed generally good agreement for detection of a “hit” (13/13 and 32/34 at the 4 and 24 hour time points).  All “hits” detected on only one plate were very close to the cut-off (mean ±3 SD).  In addition, the calculated cell percentages between plates for each compound were in close agreement.  At 4 hours, 12 compounds induced progress to early apoptosis and 2  (1 unique) to late apoptosis or death.  At 24 hours, 32 compounds induced progress to early apoptosis and 27 (1 unique) to late apoptosis or death.  Note that at the latter time point, more compounds were scored as apoptosis inducers; also, compounds which showed activity at the early time point had induced the cells to progress further through the apoptotic pathway by 24 hours.

The results for panel of 80 cytoactive compounds plus controls when tested against Jurkat cells using the Guava ViaCount assay are shown in Figure 3.  The percentages of dead and apoptotic cells were approximately 2.5% at both time points in the negative controls (n=10).  The positive control, a low concentration of sodium azide, had very little activity at either time point (n=6).  The replicate plates showed good agreement for detection of a “hit” (7/9 and 28/31 at the 4 and 24 hour time points, respectively).  All “hits” appearing on only one plate were very close to the cutoff (mean ±3 SD).  At 4 hrs, 3 compounds induced cell death and 7 (6 unique) induced apoptosis.  At 24 hours, 26 compounds induced cell death and 24 (2 unique) induced apoptosis.  Again, incubating the cells for a longer duration with cytoactive compounds allowed the detection of additional active compounds.

Figure 3: Detection of Cytotoxic Activity in a Panel of Active Compounds. Jurkat cells were cultured for either 4 hours (A, C) or 24 hours (B, D) and then analyzed for the presence of dead cells (A, B) or cells in mid-apoptosis (C, D).

Figure 3: Detection of Cytotoxic Activity in a Panel of Active Compounds. Jurkat cells were cultured for either 4 hours (A, C) or 24 hours (B, D) and then analyzed for the presence of dead cells (A, B) or cells in mid-apoptosis (C, D).

Table 1 compares the results obtained for both assays at both time points.  The Nexin assay identified more compounds with activity (13) than the ViaCount assay (8) when compounds are incubated with cells for 4 hours.  However, approximately half of the compounds (8/14) had activity in both assays at this time point.  With the longer incubation time (24 hours), most compounds (28/33) showed activity in both assay types.  Four compounds were active only in the Guava Nexin assay and one in the Guava ViaCount assay at 24 hours.

Table 1. Summary of “Hit” Compounds Results for Jurkat Cells

Table 1. Summary of “Hit” Compounds Results for Jurkat Cells

The results for the two assays correlate well because both assays identify apoptotic cells as well as live and dead cells.  The Guava Nexin assay detects an early step in programmed cell death, while the Guava ViaCount assay detects a slightly later step. The difference in the apoptotic stage detected most likely explains why the Guava Nexin assay (early apoptosis) almost always detects a higher percentage of apoptotic cells than the Guava ViaCount assay (mid-apoptosis).

To test how well the two assays can be generalized for compound screening, we screened the same panel of 80 compounds against a different type of cell line, CHO cells, an adherent Chinese Hamster Ovary fibroblast line.  Non-hematopoietic cells require longer induction periods, so CHO cells were incubated with the compounds and the controls for either 20 or 41 hours prior to assay.  Representative dot plots are shown in Figure 4.  The data for the CHO screening experiment is summarized in Table 2.

Figure 4: Examples of the Effects of “Hit” Compounds on CHO Cells. The effects of selected compounds are shown for the Guava ViaCount assay on the left and for the Guava Nexin assay on the right. Results were obtained after a 41 hour incubation with controls or test compounds.

Figure 4: Examples of the Effects of “Hit” Compounds on CHO Cells. The effects of selected compounds are shown for the Guava ViaCount assay on the left and for the Guava Nexin assay on the right. Results were obtained after a 41 hour incubation with controls or test compounds.

Table 2. Summary of “Hit” Compounds Results for CHO Cells

Table 2. Summary of “Hit” Compounds Results for CHO Cells

Figure 4 shows examples of the results obtained with CHO cells for the negative control and three test compounds assessed using the Guava ViaCount and Guava Nexin assays.  The negative control showed that the CHO cells are mostly viable.  Addition of sanguinarine sulfate, which induced mostly apoptosis and some cell death, caused a shift in the pattern of cells displayed.  In the ViaCount plot, there were now apoptotic cells which had taken up an intermediate amount of the impermeant dye and appeared between the two markers, as well as some dead cells which appeared to the right of the purple marker.  The Nexin plot shows that the apoptotic cells bound Annexin V-PE and appeared in the lower right quadrant.  The dead cells, which had also taken up the 7-AAD, now appeared in the upper right quadrant.  Acriflavium HCl induced mostly dead cells, with some apoptotic and viable cells remaining.  All three types of cells—viable, apoptotic, and dead— were seen in both assays.  Mycophenolic acid induced only a very small fraction of cells to enter apoptosis (as detected by Nexin only) and a small number progress to death (seen in both Guava ViaCount and Guava Nexin assays).

The Guava Nexin assay identified more compounds with activity (14) than the Guava ViaCount assay (10) when compounds are incubated with cells for 20 hours.  Two compounds had activity only in the Guava ViaCount assay, 6 only in the Guava Nexin assay and 8 in both assays at  this time point.  With the longer incubation time with compound (41 hours), most compounds (18/27) showed activity in both assay types.   Six compounds were active only in the Guava Nexin assay and three in the Guava ViaCount assay at 41 hours.  Again, however, the percentage of apoptotic cells detected by the Guava Nexin assay was almost always greater than that detected by the ViaCount assay.

Most compounds (23/37) induced either apoptosis or death in both cell lines (Table 3).  Ten compounds had some activity against Jurkat cells only and four compounds had some activity against CHO cells only.  Not only were more ‘hits’ detected using the Jurkat cells, but also the frequency of detecting strong apoptosis inducers was higher in Jurkats than CHO cells.  For the Nexin assay, 13 compounds induced >50% of cells to become apoptotic at one of the two time points versus only 5 for CHO cells.  In the Guava ViaCount assay, the opposite was true—no strong inducers of Jurkat cells were detected, but it did detect 3 stronger inducers of CHO cells.

Five out of 33 hits were retested against Jurkat cells to re-confirm their activity and to determine their IC50 and EC50 values.  Results replicated what was observed in the previous screening experiment.  Three of the compounds, 5-azacytidine, mebendazole, and albendazole, induced apoptotic and dead cells detectable in both assays. The apoptotic cells induced by helenine were detected only in the Guava Nexin assay but the induced dead cells were detected in both.  Ellagic acid had the greatest effect on mid-apoptotic cells stained with the Guava ViaCount Flex reagent.

Table 3. Overall comparison of Jurkat vs. CHO cells for both assays

The IC50 and EC50 values for the data shown in Figure 6 are tabulated in Table 4. In general, most compounds are equivalently potent at inducing apoptotic or dead cells.

Figure 6: Titration of “Hit” Compounds. A and B display titration data for the Guava ViaCount assay; C and D for the Guava Nexin assay. Data shown are for the 24 hour time point.

Figure 6: Titration of “Hit” Compounds. A and B display titration data for the Guava ViaCount assay; C and D for the Guava Nexin assay. Data shown are for the 24 hour time point.

Table 4. Summary of “Hit” Compound Titration

Compound ViaCount IC50 (µM) Nexin EC50 (µM)
Mid-Apoptotic Cells Dead Cells Early Apoptotic Cells Late Apoptotic & Dead Cells
5-Azacytidine 0.58 1.1 1.2 1.7
Ellagic Acid 4.1 >10 >10 >10
Mebendazole 0.15 0.16 0.22 1.6
Albendazole 0.12 0.14 0.22 0.25
Helenine >10 9.6 4.9 8.5

Discussion

The Guava ViaCount assay allows assessment of absolute cell concentration and viability of cell samples in 96-well microplates rapidly, reliably and conveniently.  The Guava Nexin assay allows the detection of cells in early apoptosis and in late apoptosis and death.  Both are mix-and-read assays, so there is very little manipulation required of the samples after the cells have been incubated with the test compounds.  Both assays also use low numbers of cells.  These assays were performed with 5 x 104 cells per well, but fewer could have been used.  Both assays are robust and yield reproducible results, either when used for screening or for quantifying the potency of compounds.

The panel of 80 compounds screened for this study contained some well known apoptosis inducers, such as camptothecin, podophyllotoxin, colchicine, azacytidine, cycloheximide, and methotrexate.  There were also many compounds, based on their mechanism of action, which should have and indeed did induce apoptosis, including sanguinarine sulfate, azathioprine, mercaptopurine, emetine, thioguanine, mechlorethamine, quinacrine, and acriflavium HCl, among others.  Upon close review of the literature, other compounds that were not expected to induce apoptosis or death based on their clinical indications were found to have activities consistent with those of other cytoactive compounds.  For example, the estrogen derivative hexestrol causes twisted ribbon-like microtubules to form19.  Aklavin HCl, originally described as an antibiotic, is also a potent antineoplastic drug, which disrupts the structure of DNA as it intercalates20.   Niclosamide, an anthelmintic, is an oxidative phosphorylation uncoupler21.  Three other anthelmintic compounds—mebendazole, fenbendazole and albedazole—each structurally related to the others, are microtubule disruptors22.  Interestingly, a fourth member of this family of anthelmintics—thiabendazole—had no apoptotic or cytotoxic activity against either Jurkat or CHO cells.  One report showed that thiabendazole apparently has a different mechanism of action from the other three and hence might not be expected to affect cells in a similar manner as other family members23.

At least one compound was determined to be a false positive, at least for apoptosis induction.  Celastrol, which appeared to strongly induce apoptosis in CHO cells and cell death in both cell lines, was found to actually dissolve the plasma membranes, leaving free nuclei in the wells.  For some reason, many of those free CHO nuclei bound Annexin V-PE and on the two color dot plots appeared to be apoptotic cells.  However, examination of the FSc versus Annexin V-PE dot plot revealed that the FSc intensity corresponded to free nuclei and not to apoptotic cells.  Thus, the FSc intensity (a rough measure of size) information provided by both the Guava ViaCount and Guava Nexin assays allows the users to determine whether the fluorescent staining is due to intact cells or not.  Another compound, ellagic acid, which is a DNA topoisomerase inhibitor and hence could induce apoptosis, showed an unusual staining pattern in the Guava ViaCount assay, inconsistent with typical apoptotic cells.  Specifically, instead of taking up more of the viability dye, the ellagic acid treated cells took up less of the membrane permeant dye and hence fell below the viability marker.  This effect was more pronounced with CHO cells.  Since both cell types did show some apoptotic cells in the Guava Nexin assay (as determined by uptake of Annexin V-PE), it is possible that the ellagic acid was inducing a little apoptosis and also having some sort of nonspecific effect on the membrane permeant dye.

There were compounds whose mechanism of action suggested that they might induce apoptosis and/or cell death but did not in this screen.  These included DNA crosslinking drugs such as cyclophosphamide, cisplatin and busulfan; intercalating agents such as chloroquine; DNA replication inhibitors such as fluoruracil; and reductase inhibitors such as hydroxyurea.  In some cases, the compounds might not have been used at a high enough concentration to be effective.  At least some well known apoptotic inhibitors are not very potent.  Etoposide has an EC50 above 10 µM and hence its activity might not have been detected in this screen had it been included in the panel.  Even camptothecin, which is considered a potent inducer, has an EC50 of only 0.2 µM.  In other cases, the compounds might not have been effective against the CHO and Jurkat cell lines used herein but could have had activity against other cell lines.  Certainly, even in this screen, we did observe cell specific effects for some of the compounds, which affected either only Jurkat cells or only CHO cells.  In addition, the compounds that theoretically should have been active, but were not in these screens, could have been insoluble in the culture medium or metabolized to inactivate derivatives of the original compounds.  Other compounds tested that were not expected to have an effect in either assay, including anti-infectives such as nitrofurazone, vadarabine, zidovudine and foscarnet.

Summary and Conclusions

The work shown here demonstrates the usefulness and reproducibility of plate based cellular assays to screen for small molecules with cytotoxic and apoptotic activity.  The Guava Nexin assay can quickly and effectively identify potent apoptotic and cytotoxic compounds.  While the Guava ViaCount assay can also detect apoptosis induction, it is more appropriate for cytotoxic compounds.  This combination of assays can identify apoptotic inducers, characterize their EC50 values and quantify the percentage of cells affected. The detection of sub-population effects for these compounds highlights the benefit of a platform capable of single cell analysis.  Because of the difference in the types of apoptotic cells detected (early versus mid for Guava Nexin and Guava ViaCount, respectively), if one is looking only for apoptotic inducers, using the Guava Nexin assay alone could suffice.  However, to better understand the mechanism of action and to gain more information about the effects of particular compounds, both assays can be performed to provide additional information.

Materials

     

References

  1. Millard, A.L., Bernard, J., et al.  Improved routine counting, mortality evaluation and phenotypical characterisation of dendritic cells by a single automatized system.  Submitted. 2004.
  2. Altman, S.A., et al. Biotechnology Progress 9:671-674, 1993
  3. Mascotti, K., et al. Transfusion 40: 693-696, 2000.
  4. Kerr, J.F.R., et al. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239-257.
  5. Wyllie, A.H., et al. Cell Death: The significance of apoptosis. Int Rev Cytol. 1980;68:251-306.
  6. Wyllie, A.H. Apoptosis. Br J Cancer. 1993;67:205-208.
  7. Majno, G., Joris, I. Apoptosis, oncosis, and necrosis: An overview of cell death. Am J Pathol. 1995;146:3-15.
  8. Rudin, C.M., Thompson, C.B. Apoptosis and disease: Regulation and clinical relevance of programmed cell death. Ann Rev Med. 1997;48:267-281.
  9. Tait, J.F., et al. Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem. 1989;264:7944-7949.
  10. Andree, H.A.M., et al. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem. 1990;265:4923-4928.
  11. van Heerde, W.L., et al. The complexity of the phospholipid binding protein Annexin V. Thrombosis and Haemostasis. 1995;73:172-179.
  12. Fadok, V.A., et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.  J Immunol. 1992;148:2207-2216.
  13. Koopman, G., et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994;84:1415-1420.
  14. Martin, S.J., et al. Early redistribution of plasma membrane phosphatidylserine in a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182:1545-1556.
  15. Vermes, I., et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Meth. 1995;184:39-51.
  16. van Engeland, M., et al. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry. 1996;24:131-139.
  17. van den Eijnde, S.M., et al. In situ detection of apoptosis during embryogenesis with Annexin V: From whole mount to ultrastructure. Cytometry. 1997;29:313-320.
  18. Schmidt, I., et al. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry. 1992;13:204-208.
  19. Chaudoreille, M.M., et al. Qualitative study of the interaction mechanism of estrogenic drugs with tubulin.  Biochem Pharmacol.  1991; 41:685-93.
  20. Yang, D. and Wang, A.H. J Biochem. 1994; 33:6595-6604.
  21. Weindbach, E.D., Garbus, J.  Mechanism of action of reagents that uncouple oxidative phosphorylation.  Nature. 1969; 221:1016-8.
  22. Pourgholami, M.H., et al. In vitro and in vivo suppression of growth of hepatocellular carcinoma cells by albendazole.  Cancer Lett.  2001; 165:43-9.
  23. Gupta, R.S. Cross-resistance of nocodazole-resistant mutants of CHO cells toward other microtubule inhibitors: similar mode of action of benzimidazole carbamate derivatives and NSC 181928 and TN-16.  Mol. Pharmacol.  1986; 30:142-8.