Flica caspase 8 assay kit

Detection of caspase-8 activity was estimated with FLICA Caspase-8 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA). Both colon adenocarcinoma cell lines (DLD-1 and HT-29) were incubated for 24 h with novel synthesized compounds, roscovitine, and 5-flurouracil at 0.5 µM concentration. The flow cytometer (BD FACSCanto II, Becton Dickinson Bioscences Systems, San Jose, CA, USA) was utilized to measure the percent of activate caspase-8 in both cell lines. Data was acquired and evaluated with FACSDiva software (ver. 6.1.3, BD Biosciences Systems, San Jose, CA, USA). The test was performed as described in the literature [48 (link)].

Caspase-8 and caspase-9 activitiesweremeasured using the FLICA Caspase-8 Assay Kit and FLICA Caspase-9 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA), respectively, according to the manufacturer’s protocol. In brief, MCF-7 and MDA-MB-231 breast cancer cells (1 × 10⁶) were washed with cold PBS twice and re-suspended in buffer. Then, 5 μL of diluted FLICA reagent and 2 μL of Hoechst 33,342 were added to 93 μL of cell suspension, mixed by pipetting and incubated for 60 min at 37 °C. After incubation, the cells were washed twice with 400 μL of apoptosis wash buffer and centrifuged at 300× g. After the last wash, the cells were resuspended in 100 μL of apoptosis wash buffer supplemented with 10 μg/mL of PI. Analyses were performed using aBD FACSCanto II flow cytometer, and the results were analyzed with FACSDiva software (version 6.1.3, BD BiosciencesSystems, San Diego, CA, USA). To identify the involvement of caspases in apoptosis induced by compounds 1–3, the investigated breast cancer cells were pretreated with the pan-caspase inhibitor, Z-VAD-FMK (100 µM) (Sigma Aldrich, St. Louis, MO, USA), as described in [28 (link)]. The activation of caspases-8 and -9 was detected as described above.