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Cell-mediated Cytotoxicity Assay: Total Cytotoxicity & Apoptosis Assay


Total Cytotoxicity & Apoptosis Assay is a single-tube, tri-color assay for quantitative assessment of cell-mediated cytolytic activity due to apoptosis and necrosis. The assay employs a green fluorescent cellular stain, CFSE, to label target cells, the red live/dead viability dye, 7-AAD, to identify the dead cells present in the cytotoxicity assay samples, and the orange-red SR-FLICA reagent, SR-VAD-FMK, to measure caspase activity in the target cell population. Analyze your results using a flow cytometer.

Catalog # Size Quantity Price
971 125 Tests $335.00
972 250 Tests $613.00
Catalog #: 971, 972 Category:

Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells. ICT’s Total Cytotoxicity Assay allows you to assess cytolytic activity in cell culture.

The Total Cytotoxicity & Apoptosis Assay includes three fluorescent reagents:  CFSE, 7-AAD, and SR-FLICA.  CFSE, a green fluorescing cellular stain, is utilized to identify the target cell population. The unstained effector cells are added and incubated with the target cells. SR-FLICA reagent is incubated with cells to identify those containing active caspases. 7-AAD, a red fluorescing live/dead stain, is then added to stain all necrotic cells red by binding to the DNA of membrane-compromised cells. Through flow cytometry, quantify four populations of cells: live, early apoptotic, late apoptotic, and necrotic.

1. Dilute 10X Assay Buffer 1:10 with diH20 and filter sterilize.
2. Prepare samples and controls (1-2 x 10^6 target cells/mL, wash, and resuspend in 1.8 mL 1X Assay Buffer.
3. Reconstitute CFSE with 200 µL DMSO and dilute 1:250 with 1X Assay Buffer, forming a 10X working solution.
4. Add 200 µL 10X CFSE to stain target cells green and incubate 15 minutes at room temperature.
5. Add cell culture media to stop CFSE binding reaction. Wash target cells and adjust cell concentration (100 µL).
6. Add effector cells (100 µL) to target cells (100 µL) and incubate 4-6 hours at 37°C.
7. Reconstitute SR-FLICA with 100 µL DMSO and dilute 1:12.6 in media, forming a 20X working solution.
8. Add 10 µL 20X SR-FLICA to label apoptotic cells orange-red and incubate 45 minutes at 37°C; wash cells. Adjust samples to 400 µL andplace cells on ice.
9. Reconstitute 7-AAD with 260 µL DMSO and dilute 1:10 with 1X Assay Buffer, forming a 21X working solution.
10. Add 20 µL 21X 7-AAD to label necrotic cells red and incubate 10 minutes on ice.
11. Run controls and analyze samples with a flow cytometer. Identify target cells by analyzing CFSE (FL-1) vs. 7-AAD (FL-3). Create a plot of SR-FLICA (FL-2) vs. 7-AAD (FL-3). CFSE excites at 492 nm and emits at 520-540 nm; SR-FLICA excites at 550-580 nm and emits at 590-600 nm; 7-AAD excites at 546 nm and emits at 647 nm.

Total Cytotoxicity & Apoptosis Assay easily distinguishes green, CFSE-labeled target cells from effector cells. Target cells are then further distinguished into living, necrotic, early apoptotic, and late apoptotic sub-populations with SR-FLICA, an orange-red poly caspase inhibitor, and 7-AAD, a red live/dead stain.

• Reagent name: 7-AAD, CFSE, SR-VAD-FMK
• Target: Cell-mediated cytotoxicity, poly caspases
• Excitation/Emission: 7-AAD – 546 nm / 647 nm; CFSE – 492 nm / 520 nm; SR-FLICA – 565 nm / 600 nm
• Method of Analysis: Flow cytometry
• Types of Samples: Cell culture
• Storage: CFSE at ≤ -20°C, other kit components at ≤2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 971: 125 Tests
• SR-FLICA Reagent (SR-VAD-FMK), 1 vial, #6221
• CFSE target cell stain, 1 vial, #6162
• 7-AAD vital stain, 1 vial, #6163
• 10X Assay Buffer, 30 mL, #6161
• Kit Manual

Kit 972: 250 Tests
• SR-FLICA Reagent (SR-VAD-FMK), 2 vials, #6221
• CFSE target cell stain, 1 vial, #6162
• 7-AAD vital stain, 2 vials, #6163
• 10X Assay Buffer, 60 mL, #685
• Kit Manual