Caspase 3 7 reagent

After treatment with SP, SB, SA, SL, BT sup, and CA sup or siRNA transfection for 48 h or BIX for 24 h, the cells were collected and incubated with the Muse Annexin V and Dead Cell Assay kit (MCH100105; Merck) for 20 min at room temperature. For analysis using the Muse™ Caspase 3/7 Kit (MCH100108; Merck), the cells were incubated with caspase 3/7 reagent (Merck) for 30 min in a humidified atmosphere with 5% CO₂ at 37 °C. After incubation, the cells were incubated with Caspase 7-AAD (Merck) for 5 min at room temperature. After incubation, ~5 × 10⁴ cells were analyzed using a Muse Cell analyzer (Merck). The FACS results were analyzed using Muse 1.6 Analysis software (Merck).

Caspase 3/7 activation was evaluated, as previously described, with minor changes [17 (link)]. 150 μL of a working solution was prepared with Caspase 3/7 reagent (Merck Millipore, Burlington, MA, USA, MCH4700-1505) mixed with PBS in 1:8 ratio. The working solution was mixed with 50 µL of cell suspension and incubated for 30 min at 37 °C. Following that, 7-AAD dye (MCH4700-1505) was diluted with Buffer BA MCH4700-1360 (1:75 ratio). 7-AAD solution, 150 µL, was mixed with THP-1 cells (5 × 10⁴), and the suspension was incubated for 5 min at RT. Early/late apoptotic cells were counted with Muse Cell Analyzer (Merck Millipore).