Caspase 3 7 assay kit

The level of caspase-3/7 activation was measured using a caspase-3/7 assay kit (Merck-Millipore, Burlington, MA, USA). After 48 h of incubation with azelastine, cells were harvested by trypsinization and incubated at 37 °C with 5 µL of Caspase-3/7 working solution (as per protocol). Then, 150 µL of Caspase 7-AAD working solution was added to the cells. Detection of caspase-positive cells was performed using a Muse analyzer (Merck-Millipore, Burlington, MA, USA).

The capacity of the Se-NSAID derivatives to induce apoptosis was analyzed using both a Caspase 3/7 assay kit and Annexin V & Dead Cell assay kit (EMD Millipore, Darmstadt, Germany). HCT-116 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well. Cells were treated either with DMSO (control) or with different concentrations of compound 5 and incubated for 24 h. At the end of the treatment, cells were collected and treated with the respective dyes according to the manufacturer’s protocol. For the Caspase 3/7 assay, cells were stained with 5 µL of Muse™ Caspase 3/7 working solution and incubated at 37 °C for 30 min. After incubation, 150 mL of Muse™ Caspase 7-AAD working solution was added and the samples were incubated for another 5 min in the dark at room temperature prior to analysis. For Annexin V assay, cells were stained with 100 µL pf Muse™ Annexin V & Dead Cell Reagent and incubated for 20 min at room temperature in the dark. Samples for both assays were analyzed on a Muse™ Cell Analyzer (Merck Millipore, Darmstadt, Germany) using Muse™ version 1.4. software.

The BICR10 cells (3 × 10⁵) were infected with lentivirus containing shRNA specific for SRSF10 gene in 6 well cell culture plates, and after 4 days, caspase activity was measured. In another set of experiment, post puromycin selection, 4 days later the cells were treated with 30 μM concentration of z-VAD-FMK pan-caspase inhibitor (Sigma, V116) after 24 h the caspase activity was measured. The caspase 3/7 activation was measured using the caspase 3/7 assay kit (Sigma, CASP3F) recommended by the manufacturer. Luminescence readings were taken using a Glomaz multi detection system (Promega).