Mhc 2 pe cy5

Freshly excised hearts were minced on ice and digested in serum free Iscoves DMEM supplemented with 2mg/mL of Collagenase Type 2 (Worthington, Cat #LS004176) and 0.02mg/mL of Deoxyribonuclease I from bovine pancreas (Sigma, Cat #DN25-100MG) at 37°C for 30 minutes. These cell suspensions were then neutralized with IDMEM containing 10% FBS and filtered through 0.45μm strainer before blocking with 2.4G2 (BD Pharmingen, Cat #553141) for 30 minutes on ice, and staining with Gr-1-APC (Clone RB6-8C5, eBioscience), CD11b-APC-Cy7 (M1/70, BD Pharmingen), Ly 6C-PerCP-Cy5.5 (HK1.4, eBioscience), Ly6G-FITC (1A8, eBioscience), MHC-II PE-Cy5 (M5/114.15.2, eBioscience), MerTK-PE (DS5MMER, eBIoscience), F4/80-PE-Cy5 (BM8, eBioscience) and CD64-Biotin (X54-5/7.1, Biolegend and used in conjunction with Streptavidin PE-Cy7, eBioscience) antibodies for 30 minutes on ice protected from light. Cells were washed with PBS prior to flow cytometry. The samples were then analyzed on BD LSR-II (UAB Flow Cytometry Core Facility). Neutrophils were identified as Gr-1⁺CD11b⁺Ly-6G⁺F4/80^-MHC-II^-. Cardiac macrophages were first gated on F4/80⁺CD11b⁺ cells and further gated for expression of MerTK and CD64. Flow cytometry data were analyzed using FlowJo software. Percent neutrophils and macrophages were determined as (Percent live gated cells from the heart) x (percent positive).

For all staining, cells were stained in PBS with 2% FCS for 20 min at 4°C. BD Aria II was used for flow sorting and BD LSRII was used to collect data. For sorted cells, debris and dead cells were excluded using FSC-SSC. Doublets were excluded using both FSC and SSC singlet gating, then CD19^+ve B cells isolated. For Figures 1C and 2A, CD4 and CD3 stains were also included to exclude contaminating T cells. Antibodies used were specific for and labeled with CD21-FITC, CD3-PE TxR, CD4-PE, IgM-APC, GM-CSF-PE, IL-10-PE, IL-17-alexa fluor647, IFN-γ-FITC (BD Biosciences); CD11b-BV570, CD11b-PE Cy5, CD19-PE, CD19-BV605, CD1d-PerCP Cy5.5, CD21/35-APC, CD23-PE, CD23-alexa fluor 647, CD23-PE Cy7, CD3-FITC, CD3 PE/Dazzle 594, CD38-PE, CD4-PE, CD4-BV605, CD40-PE Cy7, CD43-FITC, CD43-APC, CD5-PE Cy5, CD80-PerCP Cy5.5, CD86-BV421, TIM1-PE, TNFα-BV605 (Biolegend); CD21/35-APC efluor780, CD24-APC efluor780, CD25-efluor450, CD9-APC, DO11.10 TCR-Biotin, IgD-efluor450, MHCII-PE Cy5, CD19-eFluor450, F4/80-APC, IFN-γ-PE Cy7, streptavidin-PE (eBioscience); IgM-TxR, IgM-alexa fluor647 (Southern Biotech); IgM-alexa488, cell tracker green (Molecular Probes), and CFSE (fluka). All analysis was performed using FlowJo Software.