Guava Technologies, Inc.
25801 Industrial Blvd.
Hayward, CA, 94545
Website: http://www.guavatechnologies.com
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Complementary Plate-Based Cell Assays For Apoptosis And Cytotoxicity Screening
by Lisa B. English, PhD and Dianne Fishwild, PhD
Introduction
Bottlenecks in life science research often occur at the point where detailed information about cell behavior is required. Many cell-based assays report on analyses of only bulk populations. Few provide automated analysis of single cells, particularly live cells or cells in suspension. As scientists seek to understand cells in the context of "systems biology," their need for fast, easy, and accurate cell analysis methods also increases. This article will report on the use of two plate-based cell assays for flow cytometry — a technology that has become more accessible for life sciences researchers — as a successful way to detect cytotoxicity, apoptosis, or both.
Apoptosis, or programmed cell death, is an important regulatory pathway of cell growth and proliferation. Cells respond to apoptosis induction signals by initiating intracellular processes that result in characteristic physiological changes.(1-3) Early in the apoptotic pathway, phosphatidylserine (PS) is translocated to the outer surface of the cell membrane. The Guava Nexin Apoptosis Assay (Guava Technologies, Hayward, CA) uses Annexin V conjugated to phycoerythrin (PE) to monitor this externalization event as well as the dead cell dye, 7-ADD, to monitor the loss of membrane integrity that occurs in later stages of apoptosis.(4-8) Guav'a ViaCount Assay provides absolute cell counts based on the exact sampling volumes used as well as viability measurements assessed by differential permeability of DNA-binding dyes — one permeates all nucleated cells, one permeates only cells with compromised membrane integrity — thus providing more accurate and precise determinations than trypan blue.(9,10) The two assays were used to screen a panel of 80 anti-inflammatory, anti-neoplastic and anti-infective compounds for their ability to cause cell death or apoptosis induction.
Materials and methods
A non-adherent human T cell line, Jurkat, and an adherent hamster fibroblast line, CHO-K1, were kept in log phase growth in optimized media. For assays, Jurkat cells were cultured in 96-well flat bottom polystyrene plates and CHO cells were cultured in 96-well flat bottom polystyrene plates coated with hydrogel to prevent their attachment.
Approximately 5000 (cytotoxicity screening) or 50,000 (apoptosis screening) cells/well in complete medium were added to replicate 96-well plates with the compounds diluted to 10 μM and incubated at 37 C, 5% CO2 for various times. Negative controls included diluent and medium only; the positive control was 5 μM staurosporine. Assay reagents were added to the wells according to instructions, and incubations carried out at room temperature in the dark. Plates were loaded onto Guava's Microcapillary Flow Cytometry System and 1,000 (cytotoxicity) or 2,000 (apoptosis) events per well were acquired.
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Results
Figures 1-4 show examples of results obtained for the negative control and two test compounds. With negative controls, the majority of cells were viable, showing little to no cytotoxicity and apoptosis. In the viability or cytotoxicity assay, the cells absorbed the nucleated cell dye, but not the membrane impermeant dye, appearing in the upper left corner (Fig 1A, Jurkat cells; Fig 2A CHO cells). And, in the apoptosis assay, the cells bound neither Annexin V-PE nor 7-AAD, appearing in the lower left quadrant (Fig 1B, Jurkat cells; Fig 2B, CHO cells).
5-Azacytidine induced apoptosis and cell death in Jurkat cells as detected by both assays. Fig 3A shows a significant population of dead cells stained with the membrane impermeant viability dye (upper right corner). Fig 3B shows a large number of apoptotic cells stained with Annexin V-PE (lower right quadrant) as well as late apoptotic and/or dead cells stained with both Annexin V-PE and 7-AAD (upper right quadrant). While the cytotoxicity results (Fig 3A) did not clearly show apoptotic cells induced by 5-azacytidine, there was a shift in the staining pattern of viable cells, suggestive of an effect.
Addition of sanguinarine sulfate to CHO cells induced mostly apoptosis and some cell death. Fig 4A shows results from the cytotoxicity assay; apoptotic cells are labeled with an intermediate amount of the impermeant dye (between the two lines) and some dead cells (right of purple line). Fig 4B shows results from the apoptosis assay, with apoptotic cells that bound Annexin V-PE in the lower right quadrant and dead cells, which had also taken up the 7-AAD, in the upper right quadrant.
With this two-assay approach, changes in the patterns across dot plots can be used to determine the extent to which various compounds induce apoptosis and cell death.
All 80 test compounds were evaluated for potency, efficacy, and specificity. Thirty-seven compounds were found to induce apoptosis and/or cell death (i.e., hits) of which 23 exhibited minimal to substantial activity on both cell lines. Four compounds were effective against only CHO cells and 10 against only Jurkat cells, and the frequency of detecting strong apoptosis inducers was higher with Jurkat than with CHO cells.
The two assays, which both detect apoptosis induction and cytotoxicity, albeit differently, were used successfully in combination to confirm the specific activity of test compounds as well as to identify potential false positives. Both assays were robust and yielded reproducible results, either when used for screening or for quantifying potency.
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Figure 4A |
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Conclusion
The work shown here demonstrates the usefulness and reproducibility of plate-based cell assays to screen for small molecules with apoptotic and/or cytotoxic activity. The Nexin Assay identifies potent apoptotic and cytotoxic compounds. The ViaCount Assay can detect apoptosis induction, but is more appropriate for cytotoxicity screening. In combination, the assays identify apoptotic inducers, characterize EC50 values and quantify percentage of cells affected. The sub-population effects uncovered in these studies emphasizes the benefit of an approach that provides analysis at the level of single cells rather than bulk populations.
About the authors
Lisa English (lenglish@guavatechnologies.com) earned her PhD in Neuroscience from the University of Minnesota, Twin Cities, and has ten years experience in pharma/biotech corporate communications. She is currently Communications Director at Guava Technologies. Dianne Fishwild earned her PhD in Biochemistry from University of Colorado and has 20 years experience in small molecule, biologic and instrument product development. She is currently the Director of Bioengineering at Guava Technologies.
More information about plate-based cell assays is available from:
Guava Technologies
866-448-2827
www.guavatechnologies.com
References
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3. Wyllie, A.H. Apoptosis. Br J Cancer 67:205-208 (1993).
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6. Vermes, I., et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Meth 184:39-51 (1995).
7. van Engeland, M., et al. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24:131-139 (1996).
8. Schmidt, I., et al. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204-208 (1992).
9. Altman, S., et al. Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnology Progress 9:671-674 (1993)
10. Mascotti, K., et al. HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40:693-696 (2000).
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