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Cell-mediated Cytotoxicity Assay: Basic Cytotoxicity Assay


Basic Cytotoxicity Test Assay is a single-tube, dual-color assay for determining cytotoxicity by flow cytometry. The assay employs a green fluorescent cellular stain, CFSE, to label target cells and the red live/dead viability dye, 7-AAD, to identify the dead cells present in the cytotoxicity assay samples. Analyze your results using a flow cytometer.

Catalog # Size Quantity Price
969 125 Tests $155.00
970 250 Tests $258.00
Catalog #: 969, 970 Category:

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

The Basic Cytotoxicity Assay includes two fluorescent reagents:  CFSE and 7-AAD.  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. 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. The target cell and effector cell populations can then be easily distinguished, and flow cytometry easily identifies live and necrotic cells.

1. Dilute 10X Assay Buffer 1:10 with diH20 and filter sterilize.
2. Prepare sample 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 the cell concentration (200 µL).
6. Add effector cells (200 µL) to target cells (200 µL) and incubate 4-6 hours at 37°C.
7. Reconstitute 7-AAD with 260 µL DMSO and dilute 1:10 with 1X Assay Buffer, forming a 21X working solution.
8. Add 20 µL 21X 7-AAD to label necrotic cells red and incubate 10 minutes on ice.
9. Run controls and analyze sample with a flow cytometer. Identify target cells by analyzing CFSE (FL-1, green) vs. 7-AAD (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.

Basic Cytotoxicity Assay easily distinguishes green, CFSE-labeled target cells from effector cells. Target cells are then further distinguished into live and dead sub-populations with 7-AAD, a red live/dead stain.

• Reagent name: 7-AAD, CFSE
• Target: Cell-mediated cytotoxicity
• Excitation/Emission: 7-AAD – 546 nm / 647 nm; CFSE – 492 nm / 520 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 969: 125 Tests
• CFSE target cell stain, 1 vial, #6162
• 7-AAD vital stain, 1 vial, #6163
• 10X Assay Buffer, 30 mL, #6161
• Kit Manual

Kit 970: 250 Tests
• CFSE target cell stain, 1 vial, #6162
• 7-AAD vital stain, 2 vials, #6163
• 10X Assay Buffer, 60 mL, #685
• Kit Manual

Huang H, Zhang Y, Gallegos V, Sorrelle N, Zaid MM, Toombs J, Du W, Wright S, Hagopian M, Wang Z, Hosein AN, Sathe AA, Xing C, Koay EJ, Driscoll KE, Brekken RA. Targeting TGFβR2-mutant tumors exposes vulnerabilities to stromal TGFβ blockade in pancreatic cancer. EMBO Mol Med. 2019 Nov 7;11(11):e10515. doi: 10.15252/emmm.201910515. Epub 2019 Oct 14. Full Text