Live dead fixable blue stain kit

Flow cytometry was performed as previously described.^{10 (link)} Briefly, primary cardiac fibroblasts (CD105⁺CD31⁻CD45⁻) from passage 1 were cultured in 10% FBS DMEM in the absence or presence of 30 ng/mL of Neuregulin‐1β for 48 hours. Cells were trypsinized, washed with PBS and resuspended in PBS/0.5% BSA/2 mmol/L EDTA buffer containing murine Fc block reagent (BD Biosciences). The cells were then incubated with CD105‐APC (clone MJ7/19; Biolegend) antibody for 20 minutes at 4°C, washed once with 10 volumes of cold PBS/BSA/EDTA, fixed and permeabilized using 4% paraformaldehyde and 0.1% saponin in Ca/Mg free DPBS. Cells were stained with fluorescein isothiocyanate (FITC)‐conjugated anti‐αSMA (Clone 1A4, Sigma) antibody for 25 minutes at 4°C. Mouse FITC‐conjugate IgG2a (Clone UPC‐10, Sigma) were used as an isotype control. Viable and nonviable cells were distinguished using LIVE/DEAD Fixable Blue Stain kit (Invitrogen). Data acquisition was performed using an LSRII flow cytometer (BD Biosciences), and the data were analyzed with WinList 5.0 software (Verity Software House, Inc). Antigen negativity was defined as having the same fluorescent intensity as the isotype control.