Hello,

I recently performed an apoptosis analysis using the MUSE Cell Analyzer with the Annexin V-PE and 7-AAD kit. After 24 hours of treatment, my results indicated approximately 20% of cells in the early stage of apoptosis and 30% in the late stage. However, when I attempted to validate these findings through Western Blot, I was unable to detect cleaved caspase-3, even with 60 µg of protein extracted at the same time point. It's worth mentioning that my positive control, methyl methanesulfonate (MMS), has consistently worked well for both flow cytometry and Western Blot assays.

I’m trying to understand what might be causing this discrepancy between the MUSE analysis and Western Blot results. Could it be that the protein quantity in the WB is insufficient, or might there be issues with the extraction method or the detection of cleaved caspase-3? Additionally, is it possible that another form of cell death is occurring that the cytometry is identifying as apoptosis? Has anyone encountered a similar issue or have suggestions for improving the detection of cleaved caspase-3?

Thank you!