Annexin V-APC and TMRM Staining for Apoptosis

Annexin V-APC, TMRM staining: Apoptotic cell death was assessed as previously described [46 (link)]. Cells were harvested using trypsin/EDTA, resuspended in supernatant and washed in ice-cold PBS. Then, cell pellets were resuspended in 300 μL Annexin V-APC-binding buffer (PBS, 2.5 mM CaCl₂) supplemented with recombinant chicken Annexin V-APC (ImmunoTools, Friesoythe, Germany) and incubated for 10 min on ice. Subsequently, samples were analyzed using a FACS Lyric flow cytometer (Becton Dickinson, Heidelberg, Germany). To detect loss of mitochondrial membrane potential, cell pellets were resuspended in PBS supplemented with 2% FCS and 50 nM tetramethyl rhodamine (TMRM) (Merck, Darmstadt, Germany) of the potentiometric dye. Cells were incubated at 37 °C for 20 min and fluorescence was analyzed using a FACS Lyric flow cytometer. The proportion of TMRM^low and Annexin V-APC⁺ cells was calculated using FACS Suite software.