Flow Cytometric Analysis of Cardiac Cells

Flow cytometric analysis was performed using Beckman Coulter Gallios Flow Cytometer (Beckman Coulter, CA, USA). CMs were pre-stained using Cell Trace Violet (CTV) proliferation kit (Invitrogen, CA, USA) according to the manufacturer's protocol and were treated with their respective doses of NAC and DOX, as described earlier. On day 1 and day 3, the 3D spheroidal droplets with cells were cut using a blade, and cells were extracted using Miltenyi gentleMACS Dissociator (Miltenyi Biotec, MA, USA) using a Multi tissue Dissociation Kit-1 by running the Multi_B program according to the manufacturer's protocol. For 2D samples, cells were detached using trypsin-EDTA. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, then added to their assigned FACS analysis falcon tubes, and analysed using excitation and emission wavelengths of 405 and 450 nm, respectively. Negative controls included freshly isolated non-stained cells and positive controls were pre-stained with CTV.^{1,5,32 (link)}