The document describes several assays for measuring cell viability, cytotoxicity, and apoptosis. It provides details on assay mechanisms and recommended protocols for assays such as CellTiter-Glo, CellTiter 96 Aqueous, CellTiter-Blue, CellTiter-Fluor, CytoTox-Glo, CytoTox-One, Caspase-Glo 9, Caspase-Glo 8, and Caspase-Glo 3/7. Examples of dose-response and time course data generated by the assays on different cell types are also shown.

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2. CellTiter-Glo ®Assay CellTiter 96® AQ ueous One Solution CellTiter -Blue® Viability Assay CytoTox 96® Assay CytoTox -ONE™ Assay LIVE CELL PROTEASE DEAD CELL PROTEASE DEAD CELL PROTEASE LIVE CELL PROTEASE CellTiter -Fluor™ Viability Assay CytoTox-Glo ™ Assay CytoTox -Fluor™ Assay a Caspase-Glo ® 9 Assay Caspase-Glo ® 8 Assay Apo-ONE® Caspase 3/7 Assay Caspase-Glo ® 3/7 Assay Cell Viability, Cytotoxicity and Apoptosis Assays

3. 10 Minutes Add CellTiter-Glo ® Reagent Read Luminescence Reagent contains Ultra-Glo ® Luciferase, luciferin, Mg 2+ , buffer, lysis reagent and ATPase inhibitors Sensitive to as few as 10 cells ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP Luciferin Oxyluciferin + LIGHT Ultra-Glo ® Luciferase ADP a Back to Start (Cell) CellTiter-Glo ® Cell Viability Assay Cat. # G7570, G7571, G7572, G7573 See Data

4. 10 Minutes Add CellTiter-Glo ® Reagent Read Luminescence Reagent contains Ultra-Glo ® Luciferase, luciferin, Mg 2+ , buffer, lysis reagent and ATPase inhibitors Sensitive to as few as 10 cells ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP Luciferin Oxyluciferin + LIGHT Ultra-Glo ® Luciferase ADP a Back to Start (Cell) CellTiter-Glo ® Cell Viability Assay Cat. # G7570, G7571, G7572, G7573 Two-fold serial dilution of Jurkat cells in a 384-well plate. Data points represent the mean and standard deviation of 8 replicates for each cell number. Close

5. 1-4hr 37°C Add CellTiter 96 ® AQ ueous One Solution Reagent Read Absorbance 490nm Cells are not lysed so if color development is not to your liking, put back in incubator. MTS MTS formazan (soluble) PES reduced PES NADH NAD+ PES transports reducing equivalents from the interior of the cell to the exterior. a CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay Cat. # G3580, G3581, G3582 Back to Start (Cell) See Data

6. 1-4hr 37°C Add CellTiter 96 ® AQ ueous One Solution Reagent Read Absorbance 490nm Cells are not lysed so if color development is not to your liking, put back in incubator. MTS MTS formazan (soluble) PES reduced PES NADH NAD+ PES transports reducing equivalents from the interior of the cell to the exterior. a CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay Cat. # G3580, G3581, G3582 Back to Start (Cell) Effect of B9 hybridoma cell number on absorbance at 490nm measured using the CellTiter 96® AQ ueous One Solution Assay. Reagent reacted with cells for 1 hour prior to reading absorbance. Zero cell absorbance has not been substracted from the data. Close

7. 1-4hr 37°C Add CellTiter-Blue Reagent Read Fluorescence 560 Ex /590 Em Cells are not lysed so if color development is not to your liking, put back in incubator. May need optimization. Resazurin Resorufin Resazurin is reduced by metabolically active cells. Dead cells cannot reduce the compound. Reducing Enzymes a CellTiter-Blue® Cell Viability Assay Cat. # G8080, G8081, G8082 Back to Start (Cell) See Data

8. 1-4hr 37°C Add CellTiter-Blue Reagent Read Fluorescence 560 Ex /590 Em Cells are not lysed so if color development is not to your liking, put back in incubator. May need optimization. Resazurin Resorufin Resazurin is reduced by metabolically active cells. Dead cells cannot reduce the compound. Reducing Enzymes a CellTiter-Blue® Cell Viability Assay Cat. # G8080, G8081, G8082 Back to Start (Cell) Relative ability of different cell types to reduce resazurin. Serial twofold dilutions of Jurkat or HepG2 cells were prepared at 100µl/well in a 96-well plate andcultured for 1.5 hours at 37°C. CellTiter-Blue® Reagent (20µl/well) was added and cells were incubated for 1 hour before recording fluorescence (560 Ex/ 590 Em ) Close

9. GF-AFC GF + AFC lcp lcp The peptide substrate Gly-Phe-AFC crosses the membrane and “Live Cell” Protease (lcp) within intact cells cleaves GF-AFC. Add 100µl of CellTiter-Fluor™ Reagent 0.5-3 hours “Live Cell” Protease does not function outside cell a Back to Start (Cell) Read Fluorescence Live: 400 Ex /505 Em CellTiter-Fluor ™ Cell Viability Assay Cat. # G6080, G6081, and G6082 See Data

10. GF-AFC GF + AFC lcp lcp The peptide substrate Gly-Phe-AFC crosses the membran and “Live Cell” Protease within intact cells cleaves GF-AFC. Add 100µl of CellTiter-Fluor™ Reagent 0.5-3 hours “Live Cell” Protease does not function outside cell a Back to Start (Cell) Read Fluorescence Live: 400 Ex /505 Em CellTiter-Fluor ™ Cell Viability Assay Cat. # G6080, G6081, and G6082 Serial dilutions of Jurkat cells were plated. Half received no treatment (viable) and the other half were treated to induce cytotoxicity (treated). CellTiter-Fluor reagent was added and incubated for 30 minutes at 37°C and fluorescence read (400 Ex /505 Em ). Close

11. INT INT Formazan Lactate + NAD + Pyruvate + NADH Diaphorase transfer 50µl of conditioned culture media Add 50µl of Substrate Mix (mix just prior to use) Read Absorbance at 490nm 30 minutes Add 50µl of Stop Solution You can assay the cultured cells for viability, caspase activity, etc. after you remove the conditioned media for assay. a Back to Start (Cell) LDH LDH CytoTox 96 ® Non-Radioactive Cytotoxicity Assay Cat. # G1780 See Data

12. INT INT Formazan Lactate + NAD + Pyruvate + NADH Diaphorase transfer 50µl of conditioned culture media Add 50µl of Substrate Mix (mix just prior to use) Read Absorbance at 490nm 30 minutes Add 50µl of Stop Solution You can assay the cultured cells for viability, caspase activity, etc. after you remove the conditioned media for assay. a Back to Start (Cell) LDH LDH CytoTox 96 ® Non-Radioactive Cytotoxicity Assay Cat. # G1780 Human HepG2 cells were plated at 40,000 cells per well and allowed to grow to confluency. Cells were treated for 24 hours with the indicated concentration of staurosporine before LDH release was measured with the CytoTox 96® Assay. Data were corrected for vehicle-only control values. Close

13. Resazurin Resorufin Lactate + NAD + Pyruvate + NADH Diaphorase Add 100µl of CytoTox-One™ Reagent Read Fluorescence 560 Ex /590 Em 10 minutes Add 50µl Stop Solution Also works if you take off a portion of the conditioned media to a new plate and perform the assay. Allows multiplexing. a Back to Start (Cell) LDH LDH CytoTox-ONE™Homogeneous Membrane Integrity Assay Cat. # G7890, G7891, G7892 See Data

14. Resazurin Resorufin Lactate + NAD + Pyruvate + NADH Diaphorase Add 100µl of CytoTox-One™ Reagent Read Fluorescence 560 Ex /590 Em 10 minutes Add 50µl Stop Solution Also works if you take off a portion of the conditioned media to a new plate and perform the assay. Allows multiplexing. a CytoTox-ONE™Homogeneous Membrane Integrity Assay Cat. # G7890, G7891, G7892 Back to Start (Cell) LDH LDH Staurosporine dose response curve. Confluent HepG2 cells were treated for 24 hours with staurosporine from 48nM to 25μM to generate a dose response curve. %CVs were below 5% for all drug concentrations showing the robustness of both the CytoTox-ONE™ Assay and the robotic platform. Close

15. AAF-Luciferin AAF + Luciferin Add 100µl of CytoTox-Glo™ Reagent 15 Minutes “ dead cell” Protease released from cells with compromised membranes UltraGlo ® Luciferase + ATP LIGHT The Ala-Ala-Phe-Luciferin cannot cross intact cell membranes a Back to Start (Cell) dcp dcp Read Luminescence CytoTox-Glo™ Cytotoxicity Assay Cat. # G9290, G9291, and G9292 See Data

16. AAF-Luciferin AAF + Luciferin Add 100µl of CytoTox-Glo™ Reagent 15 Minutes “ dead cell” Protease released from cells with compromised membranes UltraGlo ® Luciferase + ATP LIGHT The Ala-Ala-Phe-Luciferin cannot cross intact cell membranes a Back to Start (Cell) dcp dcp Read Luminescence CytoTox-Glo™ Cytotoxicity Assay Cat. # G9290, G9291, and G9292 Comparison of the CytoTox-Glo™ Assay to a fluorescent LDH assay. Jurkat cells were treated with digitonin at 30µg/ml to induce cytotoxicity. Close

17. AAF-Rhodamine110 AAF + Rhodamine 110 Add 100µl of CytoTox-Fluor™ Reagent 0.5-3 hours “ dead cell” Protease released from cells with compromised membranes Can be multiplexed with Luminescent Caspase assays. The Ala-Ala-Phe-RHO110 cannot cross intact cell membranes a Back to Start (Cell) dcp dcp Read Fluorescence 485 Ex /520 Em CytoTox-Fluor™ Cytotoxicity Assay Cat. # G9260, G9261, and G9262 See Data

18. AAF-Rhodamine110 AAF + Rhodamine 110 Add 100µl of CytoTox-Fluor™ Reagent 0.5-3 hours “ dead cell” Protease released from cells with compromised membranes Can be multiplexed with Luminescent Caspase assays. The Ala-Ala-Phe-RHO110 cannot cross intact cell membranes a Back to Start (Cell) dcp dcp Read Fluorescence 485 Ex /520 Em CytoTox-Fluor™ Cytotoxicity Assay Cat. # G9260, G9261, and G9262 CytoTox-Fluor Assay multiplexed with Caspase-Glo® 3/7 Assay. LN-18 cells were plated at 10,000 cells/well and allowed to attach overnight. Cells were treated with staurosporine for 6 hours prior to assay. The CytoTox-Fluor Assay Reagent is added to wells and cytotoxicity measured after incubation for 30 minutes at 37°C. Caspase-Glo® 3/7 Reagent is added and luminescence measured after a 30-minute incubation. Close

19. proCaspase 9 Caspase 9 LEHD-Luciferin LEHD + Luciferin Add 100µl of Caspase-Glo ® 9 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 9 activity. MG-132 inhibitor present to inhibit caspase-like proteasome activity a Caspase-Glo ® 9 Assay Cat. # G8210, G8211, G8212 Back to Start (Cell) See Data

20. proCsp 9 Csp 9 LEHD-Luciferin LEHD + Luciferin Add 100µl of Caspase-Glo ® 9 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 9 activity. MG-132 inhibitor present to inhibit caspase-like proteasome activity a Caspase-Glo ® 9 Assay Cat. # G8210, G8211, G8212 Back to Start (Cell) Inclusion of proteasome inhibitor MG-132 improves Caspase-Glo 9 Assay data quality and confidence. Jurkat cells were plated at 25,000 cells per well in a 96 well plate. Apoptosis was induced for 3 hours with 5µM staurosporine or vehicle alone. Cells were assayed with Caspase-Glo 9 reagent with or without MG-132. Close

21. proCaspase 8 Caspase 8 LETD-Luciferin LETD + Luciferin Add 100µl of Caspase-Glo ® 8 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 8 activity. MG-132 inhibitor present to inhibit caspase-like proteasome activity a Caspase-Glo ® 8 Assay Cat. # G8200, G8201, G8202 Back to Start (Cell) See Data

22. proCsp 8 Csp 8 LETD-Luciferin LETD + Luciferin Add 100µl of Caspase-Glo ® 8 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 8 activity. MG-132 inhibitor present to inhibit caspase-like proteasome activity a Caspase-Glo ® 8 Assay Cat. # G8200, G8201, G8202 Back to Start (Cell) Inclusion of proteasome inhibitor MG-132 improves Caspase-Glo 8 Assay data quality and confidence. U937 cells were plated at 15,000 cells per well in a 96 well plate. Apoptosis was induced for 5 hours with 100ng/well soluble recombinant TRAIL (TNF-related apoptosis-inducing ligand) or vehicle alone. Cells were assayed with Caspase-Glo 8 reagent with or without MG-132. Close

23. proCaspase 3/7 Caspase 3/7 DEVD-Luciferin DEVD + Luciferin Add 100µl of Caspase-Glo ® 3/7 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 3 or Caspase 7 activity a Caspase-Glo ® 3/7 Assay Cat. # G8090, G8091, G8092 and G8093 Back to Start (Cell) See Data

24. proCsp 3/7 Csp 3/7 DEVD-Luciferin DEVD + Luciferin Add 100µl of Caspase-Glo ® 3/7 Reagent Read Luminescence 0.5-3 hours UltraGlo ® Luciferase + ATP LIGHT Reagent lyses cells and measures active Caspase 3 or Caspase 7 activity a Caspase-Glo ® 3/7 Assay Cat. # G8090, G8091, G8092 and G8093 Back to Start (Cell) The Caspase-Glo ® 3/7 Assay produces luminescence that is linear over a broad range of cell numbers. Jurkat cells were treated with anti-Fas mAb for 4.5 hours to induce apoptosis or were left untreated. Caspase-Glo ® 3/7 Reagent was added directly to the cells in 96-well plates and incubated for 1 hour before recording luminescence. Each point represents the average of 4 wells. The "no cell" blank control value has been substracted from each. Close

25. proCaspase 3/7 Caspase 3/7 Rhodamine 110-(DEVD) 2 2 DEVD + Rhodamine 110 Add 100µl of Apo-ONE Reagent 0.5-18 hours Reagent lyses cells and measures active Caspase 3 or Caspase 7 activity a Apo-ONE ® Homogeneous Caspase 3/7 Assay Cat. # G7790, G7791, G7792 Back to Start (Cell) Read Fluorescence 485 Ex /520 Em See Data

26. proCsp 3/7 Csp 3/7 Rhodamine 110-(DEVD) 2 2 DEVD + Rhodamine 110 Add 100µl of Apo-ONE Reagent 0.5-18 hours Reagent lyses cells and measures active Caspase 3 or Caspase 7 activity a Apo-ONE ® Homogeneous Caspase 3/7 Assay Cat. # G7790, G7791, G7792 Back to Start (Cell) Read Fluorescence 485 Ex /520 Em Increased sensitivity with Apo-ONE Caspase 3/7 Assay. Apoptosis was induced in Jurkat cells by 5 hour anti-Fas receptor antibody treatment. Close