Cell-mediated Cytotoxicity Assay: Total Cytotoxicity Test

1. Prepare samples and controls, 2. Dilute Assay Buffer 1:10 with diH2O and filter sterilize., 3. Reconstitute CFSE with 200 µL DMSO and dilute 1:250 with 1X Assay Buffer., 4. Add 200 µL CFSE to target cells to stain them green and incubate 15 minutes at room temperature., 5. Wash target cells and adjust cell concentration., 6. Add effector cells to target cells and incubate 4-6 hours., 7. Reconstitute SR-FLICA with 100 µL DMSO and dilute 1:12.6., 8. Add 10 µL SR-FLICA to label apoptotic cells orange-red and incubate 45 minutes at 37°C., 9. Reconstitute 7-AAD with 260 µL DMSO and dilute 1:10 with 1X Assay Buffer., 10. Add 20 µL 7-AAD to label necrotic cells red and incubate 10 minutes on ice., 11. Run controls and analyze with a flow cytometer. Identify target cells by analyzing FL-1 (green) vs. FL-3 (red). Create a plot of FL-2 (orange-red) vs. FL-3 (red). CFSE excites at 492 nm and emits at 520-540 nm; 7-AAD excites at 546 nm and emits at 647 nm; SR-FLICA excites at 565 nm and emits at 600 nm.