Bcl 2 activation dual detection assay

The flow cytometric analyses used in this study are Annexin V & Dead Cell assay, Multicaspase assay, Bcl-2 Activation Dual Detection assay, and Cell Cycle Analysis (MCH100105, MCH100109, MCH200105, and MCH100106) from EMD Millipore. Assays were performed as per protocols published earlier [19 (link)]. Cells were plated at 2 × 10⁵ cells per well in triplicate in 12-well culture plates. Control groups received the growth media, and for the treatment group, 5 μM of casticin in growth media was added. After 48 h of incubation, the cytometric analyses were performed with a Muse cell analyzer as per the manufacturer’s instructions.

For the various flow cytometric analyses, 1 × 10⁵ cells per well were seeded in triplicate in 6-well cell culture plates and treated with 5 μM of 4,5-diCQA for 72 hours. Cells were then harvested and stained as per the MUSE cell kit protocols. The various flow cytometric analyses performed are MUSE Annexin V & Dead cell assay (MCH100105), Mitopotential assay (MCH100110), Bcl2 Activation Dual detection assay (MCH200105), PI3K-MAPK Dual Activation detection assay (MCH200108), and Cell Cycle analysis (MCH100106) (EMD Millipore). The cells were analyzed as per the manufacturer's instructions provided in the kit (assay kit reference numbers provided in parenthesis above) using a MUSE cell analyzer. Each experiment was done at least 3 times independently.