Flow Cytometry Analysis of Cardiac Microtissues

Flow cytometry was conducted in accordance with our previous study with modifications^{24 (link)}. Cardiac microtissues were dissociated by incubation with Accumax and stained with one or a combination of the following surface markers: anti-PDGFRβ conjugated with phycoerythrin (PE), clone 28d4, 1:100 (BD, Franklin Lakes, NJ, USA), and anti-VE-cadherin conjugated with allophycocyanin (APC), clone 55-7h1, 1:100 (BD). To eliminate dead cells, cells were stained with the LIVE/DEAD fixable Aqua dead cell staining kit (Thermo Fisher). For cell surface markers, staining was carried out in PBS with 5% FBS. For intracellular proteins, staining was carried out in cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of troponin T (cTnT) (clone 13-11) (Thermo Fisher) labelled with APC using Zenon technology (Thermo Fisher) (1:50). The staining was performed in PBS with 5% FBS and 0.75% saponin (Sigma). The stained cells were analysed by a BD FACS Aria II (BD) or CytoFLEX S (Beckman Coulter, Brea, CA, USA). Data were collected from at least 10,000 events. Data were analysed with DIVA software (BD) or CytExpert software (Beckman Coulter).