Caspase3/7ActFCKit, Grn, 100tests

A flow cytometry-based assay from STEMCELL Technologies for detecting active caspase-3 in mammalian cells undergoing apoptosis. Caspases are proteases central to apoptosis, cleaving select proteins and disassembling the cell. Caspase-3 has substrate selectivity for the Asp-Glu-Val-Asp (DEVD) peptide sequence. The kit supplies a cell-permeable, non-toxic fluorogenic caspase-3/7 substrate (TF2-DEVD-FMK), an assay buffer, and propidium iodide. When added to apoptotic cells, TF2-DEVD-FMK irreversibly binds activated caspase-3, retaining the green fluorescent reagent within cells for flow cytometry detection. The kit screens caspase-3 inhibitors and quantifies apoptotic cells containing active caspase-3.

The substrate has an excitation wavelength of 503 nm, an emission wavelength of 525 nm, and an extinction coefficient of 75,000 cm⁻¹M⁻¹, supplied as a liquid. The kit provides TF2-DEVD-FMK (500X) at 100 µL, Propidium Iodide (500X) at 100 µL, and Assay Buffer at 50 mL, all stored at -20°C.

The staining protocol handles 2.5 x 10⁵ to 5 x 10⁵ cells in 0.5 mL of culture medium or buffer per sample, with incubation at 37°C and 5% CO₂ for 1 - 4 hours. After washing, fluorescence is read on a flow cytometer with a 530/30 nm filter (FITC channel). For research use only; not intended for human or animal diagnostic or therapeutic uses.