Cyan adp lx nine colour analyser

H9c2 cells were treated with FIX & PERM reagents (ADG Wien, Austria) and then stained for 30 min at room temperature, in the dark, with the anti‐γH2AX (Ser139) rabbit antibody (9718, Cell Signalling, Leiden, Netherlands, RRID:AB_2118009) and anti‐cleaved caspase‐3 rabbit antibody (559565, BD Pharmingen, San Diego, CA, USA, RRID:AB_397274). Then secondary antibody incubation was performed with the anti‐rabbit IgG (H + L) APC (4050‐11S, Southern Biotech, Birmingham, AL, USA, RRID:AB_2795959) for 30 min at room temperature, in the dark. Samples were analysed on a CyAn™ ADP LX nine‐colour analyser (Beckman Coulter, Brea, CA, USA).

We quantified proliferation activity by measuring the proportion of cells that had incorporated EdU during the S phase of the cell cycle. Two hours after EdU incorporation (10 µM), cells were subjected to an aldehyde fixation and a detergent permeabilization protocol. EdU was detected after inducing a copper-catalysed covalent reaction between an azide group coupled to Alexa Fluor ® 488 dye and an alkyne present in the ethynyl EdU moiety. This process was achieved after a 30 min incubation at room temperature in the dark on a seesaw rocker with a cocktail of Click-iT ® EdU Flow Cytometry Assay Kit reagents (Invitrogen Corporation, Carlsbad, CA) following the manufacturer’s instructions. Finally, the cells stained with FxCycle™ Violet Stain (Invitrogen Corporation, Carlsbad, CA), diluted 1:500 in 1X Click-iT ® saponin-based buffer, was analysed to quantify the total DNA content. Sample cells were sorted using a low flow rate on a CyAn ADP LX nine-colour analyser (Beckman Coulter, Brea, CA). Data were analysed using Summit 4.3 software (Beckman Coulter, Brea, CA).