Cell-mediated Cytotoxicity Assay: Total Cytotoxicity Test
The Total Cytotoxicity Test Assay Kit 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.
Cell-mediated Cytotoxicity Assay: Total Cytotoxicity Test
Cytolytic activity is an important process for eliminated intracellular pathogens and cancer cells. ICT’s Total Cytotoxicity Assay Kit allows you to assess cytolytic activity in cell culture.
The Total Cytotoxicity Assay Kit includes three fluorescent reagents: CFSE, 7-AAD, and SR-FLICA. CFSE, a green fluorescing membrane stain, is utilized to identify the target cell population. The unstained effector cells are added and incubated with the target cells. SR-LFICA 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. This further cellular differentiation in the Total Cytotoxicity Kit leads to a more accurate assessment of cytolytic activity.
7-AAD, CFSE, SR-VAD-FMK
Cell-mediated cytotoxicity, poly caspases
7-AAD – 546 nm / 647 nm
CFSE – 492 nm / 520 nm
SR-FLICA – 565 nm / 600 nm
Flow cytometry
Cell culture
CFSE at ≤ -20°C, other components at 2-8°C
Domestic: Overnight Delivery; International: Priority Shipping
United States
- Prepare samples and controls
- Dilute Assay Buffer 1:10 with diH2O and filter sterilize.
- Reconstitute CFSE with 200 µL DMSO and dilute 1:250 with 1X Assay Buffer.
- Add 200 µL CFSE to target cells to stain them green and incubate 15 minutes at room temperature.
- Wash target cells and adjust cell concentration.
- Add effector cells to target cells and incubate 4-6 hours.
- Reconstitute SR-FLICA with 100 µL DMSO and dilute 1:12.6.
- Add 10 µL SR-FLICA to label apoptotic cells orange-red and incubate 45 minutes at 37°C.
- Reconstitute 7-AAD with 260 µL DMSO and dilute 1:10 with 1X Assay Buffer.
- Add 20 µL 7-AAD to label necrotic cells red and incubate 10 minutes on ice.
- 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.
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