The Basic Cytotoxicity Test Assay Kit is a single-tube, dual-color assay for determining cell toxicity 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.
- 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.
- 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). CFSE excites at 492 nm and emits at 520-540 nm; 7-AAD excites at 546 nm and emits at 647 nm.
Product Specific References
|37120009||Lee, M.H., et al. 2023. Live Mycobacterium paragordonae induces heterologous immunity of natural killer cells by eliciting type I interferons from dendritic cells via STING-dependent sensing of cyclic-di-GMP. Microbes and infection, 105144.|