Caspase glo 3 7 3d assay kit

Cell viability and caspase-3/7 activities were evaluated using the CellTiter-Glo® 2.0 Cell Viability Assay kit (Promega, Madison, WI, USA) and Caspase-Glo® 3/7 3D Assay kit (Promega, Madison, WI, USA), respectively, according to the manufacturer’s instructions. H1975, PC9, H3122, and HC827 cells were plated at a density of 1.0 × 10⁴ cells/well in 50 µL of complete culture medium into 96-well plates. Then, 50 µL of each drug was added at the indicated final concentration (i.e., 0.1, 1.0, 10, and 100 µM). After 6 h, 100 µL of the Viability reagent were added to all wells. The samples were then mixed with an orbital shaker for 2 minutes and incubated at room temperature for 10 min. Luminescence was measured for viability using a plate reader (Enspire; PerkinElmer, Waltham, MA, USA). Next, H1975, PC9, H3122, and HC827 cells were plated at a density of 5.0 × 10³ cells/well in 10 µL of complete culture medium into 384-well plates. Then, 10 µL of each of the drugs were added at the specified final concentration. After 6 h, 20 µL of the Viability reagent were added to all wells. The samples were then mixed with an orbital shaker at 500 rpm for 30 s, and incubated at room temperature for 30 min. The caspase activation was determined by measuring the luminescence with an Enspire instrument.

The potential impact of psilocybin on the reduction in Caspase 3 and Caspase 7 activity in the β-cells exposed to HG-HL conditions was evaluated using the Caspase-Glo^® 3/7 3D Assay kit (Cat: G8981) obtained from Promega Corporation (Madison, WI, USA). β-cells were plated at a concentration of 5 × 10⁴ cells per well in a 96-well plate. After 48 h, the samples were exposed to psilocybin for 2 h. Afterwards, the cells were subjected to a medium containing psilocybin and/or HG-HL for the subsequent 48 h. Upon completion of the incubation phase, the plate was taken out and allowed to reach room temperature before adding Caspase-Glo^® 3/7 3D Reagent to each well. The contents were thoroughly mixed and incubated for a minimum of 30 min before measuring luminescence using a plate-reading luminometer (SpectraMax i3x Multi-Mode Microplate Reader, Molecular Devices, San Jose, CA, USA).