Caspglow fluorescein active caspase 8 and 9 staining kit

Caspase 8 and 9 activities were detected using the CaspGLOW™ Fluorescein Active Caspase-8 and −9 Staining Kit, respectively (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Briefly, HaCaT cells were seeded in 96-well plates at a concentration of 6.0 × 10³ cells/well and treated with titanium dioxide for six hours. Cells were then trypsinized and 300 µL of each sample was placed in Eppendorf tubes. One microliter of FITC-LEHD-FMK was added to each tube and incubated for 30 min at 37 °C. Cells were then centrifuged at 3000 rpm for five minutes and the supernatant was removed. Samples were then resuspended in Wash Buffer before being centrifuged as above. For analysis, samples were resuspended in 100 µL Wash Buffer and then plated on a black microtiter plate. Fluorescence intensity was measured at 485/535 nm. Wells containing unlabeled cells were used as control. The caspase inhibitor Z-VAD-FMK (1 µL/mL) was used as a negative control by pre-treating cells for one hour to inhibit caspase activation.

Caspase 8 and 9 activities were detected using the CaspGLOW™ Fluorescein Active Caspase-8 and -9 Staining Kit respectively (BioVision) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a concentration of 6.0 × 10³ cells/well, and treated for 6 h. Trypsinized cells were centrifuged at 3000 rpm for 5 min and the supernatant was removed before resuspension in Wash Buffer for additional centrifugation as above. Fluorescence intensity was measured at 485/535 nm using a Gen 5 2.0 All-In-One Microplate Reader (BioTek Instruments Inc.). Wells containing unlabeled cells were used for control. The caspase inhibitor Z-VAD-FMK (1 μl/ml) was used as a negative control by pre-treating cells for 1 h to inhibit caspase activation.