Gp38 apc

Cells in single cell suspensions were blocked with anti-mouse CD16/CD32 (1:200; Clone 93, Thermo Fisher Scientific) for 10 min at 4°C, followed by incubation on ice for 30 min with the appropriate combination of fluorochrome-conjugated antibodies diluted in FACS buffer: TER119-PE, (1:600, clone TER-11, eBioscience), CD45-PE (1:300, clone 30-F11, eBioscience), CD31-PE (1:300, clone 390, Thermo Fisher Scientific), gp38-APC (1:100, clone 8.1.1, eBioscience), CD90.2-eFluor 450 (1:300, clone 53-2.1, eBioscience), Sca-1- PerCP-Cy5.5 (1:300, clone D7, eBioscience), CD44-PE-Cy7 (1:300, clone IM7, eBioscience), CD140a-PE-Cy7 (1:300, clone APA5, eBioscience). 4′,6-diamidino-2-phenylindole (DAPI 1 μg/mL, Roche) was used to distinguish live/dead cells. Cells were analyzed with the BD LSR Fortessa flow cytometer (BD Biosciences) and sorted with the FACSAria III 4L (BD Biosciences). FlowJo (version 10.08) was used for data analyses.

Mouse hearts were collected and enzymatically dissociated to obtain a single-cell suspension for flow cytometry analysis. The cell pellet was resuspended in FACS buffer (PBS supplemented with 1% FBS and 1 mM EDTA) to obtain a single-cell suspension and subsequently blocked with anti–mouse CD16/CD32 (1:200; Thermo Fisher Scientific, 14-0161-82) for 10 minutes at 4°C. The cells were then incubated on ice for 30 minutes with a suitable combination of fluorochrome-conjugated antibodies diluted in FACS buffer: Ter119 PE (eBioscience, 11-5921-82), CD45 PE (1:300, eBioscience, 14-0451-82), CD31 PE (1:300, Thermo Fisher Scientific, 14-0311-82), and gp38 APC (eBioscience, 14-5381-82). Live/dead cells were distinguished using DAPI (1 μg/mL). Sorted lineage^– (Ter119⁻CD45⁻CD31⁻) and gp38⁺ cells were cultured and induced to differentiate with 10 ng/mL mouse recombinant TGF-β1 (BioLegend, 763104) (66 (link)).

Two-color flow cytometry (Lin⁻:Ter119⁻CD45⁻CD31⁻, GP38⁺) was used to identify cardiac fibroblasts from the adult mouse heart as previously described^{20 (link)}. Briefly, the hearts in the sham and MI groups were perfused with langendorff fluid for 5 min and digested with a mixture of trypsin and collagenase for 15 min. The hearts were cut into small pieces and the digestion was terminated using the serum. These tissues were filtered through a 10-μm cell strainer followed by centrifugation, and the supernatants were washed with phosphate buffer saline (PBS). Single-cell suspension was obtained by resuspending the cell pellet in PBS buffer. Cells were incubated for 30 min with the appropriate fluorochrome-labeled antibodies (Ter119-PE, CD45-PE, CD31-PE, and GP38-APC [eBioscience, USA]). The cells were analyzed using a flow cytometer. Three independent experiments were performed.