Cytoflex s

The cardiac fibroblasts were harvested using Accutase (Millipore, Billerica, MA, USA) and resuspended in flow cytometric staining buffer (1% FBS + 0.1% NaN₃ in PBS) without fixation. Flow cytometric analysis of endothelial cell surface marker was performed using CD31-Brilliant Violet 421 (102423, Biolegend, San Diego, CA, USA) antibody by a Beckman-Coulter (Dako) CytoFLEX S. Data was analyzed using FloJo software.

Flow cytometric analysis of cell surface markers was performed using CD31-Brilliant Violet 421 (102423, Biolegend, San Diego, CA, USA) and isolectin B4 (B-1205, Vector Labs) antibodies. For isolectin B4, cell fixation and permeability were performed following the protocol of the BD flow kit and DyLight 488 Streptavidin was used as the secondary antibody (SA-5488, Vector Lab). Non-myocytes were isolated from the heart using the fibroblast isolation protocol but the cells were seeded in fibronectin-coated culture dishes overnight. After 24 h minimum culture, the non-myocytes were dissociated using Accutase (Millipore, Billerica, MA, USA) and immunostained with flow cytometric staining buffer (1% FBS + 0.1% NaN₃ in PBS) for 30 min at 4 °C, followed by washing with the washing buffer and subsequent analysis using a Beckman-Coulter (Dako) CytoFLEX S. The data obtained were analyzed and presented using Flowjo software. The negative control group was processed without primary antibody first and subjected to all other processes the experimental groups were exposed to. Flow cytometric sorting for RNA-sequencing (RNA-seq) was immediately performed after isolating non-myocytes using the BD Influx (BD Biosciences, CA, USA).