Fam flica caspase assay kit

Activation of the caspase cascade occurs as a result of the initiation of the apoptotic process in the cell and is induced by the cytotoxic activity of the compound. In this regard, assessment of initiator (caspases 8, 9, and 10) and executioner (caspases 3 and 7) caspase activity was performed using FAM-FLICA^® Caspase Assay kits (all from ImmunoChemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. After 24 h incubation of MCF-7 and MDA-MB-231, breast cancer cells with the tested compounds (concentrations of 1.5 and 3 µM), the cells were collected, washed twice with cold PBS, and resuspended in Apoptosis Wash Buffer to a final concentration of 5 × 10⁵ cells/mL. In the next step, 290 µL each of cell suspension was taken and transferred into tubes. Then, 10 µL of FLICA solution diluted immediately before use (1:5 v/v, using PBS) was added to the cells, mixed by pipetting, and incubated in the dark for 1 h at 37 °C. After this time, the cells were washed twice with 2 mL Apoptosis Wash Buffer, centrifuged, and resuspended in 300 µL of the buffer. Thus, prepared samples were immediately analyzed using a BD FACSCanto II flow cytometer (10,000 events) with FACSDiva software (both from BD Biosciences Systems, San Jose, CA, USA). The equipment calibration was performed using BD Cytometer Setup and Tracking Beads (BD Biosciences, San Diego, CA, USA).