Pe cy7 conjugated anti cd31

Briefly, at most 8 × 10⁶ CD4⁺-T-cells were surface stained with 5 µL peridinin-chlorophyll-protein-Cy5-5 (PerCpCy5.5)-conjugated anti-CD127 (eBioscience, Frankfurt, Germany), 20 µL phycoerythrin (PE)-conjugated anti-ICOS (BD Biosciences, Heidelberg, Germany), 5µL allophycocyanin-H7 (APC-H7)-conjugated anti-CD45RA (BD Biosciences), and 5 µL phycoerythrin-cyanine 7 (PE-Cy7)-conjugated anti-CD31 (eBioscience) mouse monoclonal antibodies. Intracellular staining for the detection of FoxP3 was performed using a fluoresceinisothiocyanat (FITC)-conjugated anti-human FoxP3 staining set (clone PCH101, eBioscience) according to the manufacturer’s instructions. Detection of Ki67⁺ cells within the different Treg/Tresp subsets was performed by incubating the fixed cells with 2 µL Alexa-flour 647-conjugated anti-Ki67-conjugated mouse monoclonal antibodies (clone B56, BD Biosciences). Negative control samples were incubated with isotype-matched antibodies. FSC-H versus FSC-A and FSC versus SSC gating was used for doublet and debris discrimination (for gating strategy see Figure S1). Cells were analyzed by a FACS Canto flow cytometer (BD Biosciences). Statistical analysis was based on at least 100,000 CD4⁺-T-cells.

Cardiac fibroblasts (PDGFR α⁺) and endothelial cells (CD31 ⁺) were isolated from wild-type C57Bl/6J mouse hearts and sorted using APC-conjugated anti-PDGFR α⁺ (eBioscience 17-1401-81) and PE-CY7-conjugated anti-CD31 (eBioscience 25-0311-82) antibodies as described previously [37 (link)]. Cardiomyocytes were isolated from wild-type C57Bl/6J mice as described previously [53 (link)].