Cd11b apc cy7

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).

Fluorescein-conjugated ShK-F6CA was purchased from Peptides International (Louisville, KY) [17 (link), 18 (link)]. Fluorophore-conjugated antibodies for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PE-Cy7, CD45-FITC, Ly6c-PE) [13 (link)]. Polystyrene phycoerythrin-fluorescent 1-μm microspheres (Thermo Fisher #F13083) were used for phagocytosis assays [16 (link)]. Percoll was purchased from Sigma-Aldrich (#P1644).

It was performed after collagenase digestion and an additional wash with 10 mM EDTA to ensure the separation of lipid-laden macrophages, as we previously described (Nov et al. 2013 (link), Shapiro et al. 2013 (link)). After FcBlock (BD Biosciences, Franklin Lakes, NJ, USA), cells were stained with the following conjugated antibodies (10 min on ice in the darkness): CD45-APC, F4/80-PE-Cy7 (both from E-Bioscience, San Diego, CA, USA) and CD11b-APC-Cy7 (BD Pharmingen, San Diego, CA, USA). Cells were washed and pellets were then stained for 20 min on ice with BODIPY 493/503 (3 μg/mL BODIPY for 5 × 10⁶ cells; Invitrogen, D3922). Stained samples were further washed and filtered using 100 μm mesh. Propidium iodide (0.2 μg/mL; Sigma, P4864) was added to all samples. Stained samples were analyzed by FACS (Canto, BD Biosciences).

Following the centrifugation of BAL fluid to isolate leukocytes, a total cell count was performed using a Neubauer Haemocytometer and trypan blue to exclude dead cells. Samples were then treated with anti-CD16/32 (BD Pharmingen) and stained at 4 °C for 25 min with the following pre conjugated mAbs against the indicated antigens: MHCII-FITC, NK1.1-FITC, CD4-PerCP Cy 5.5, CD11c-PECy7, B220-PE Texas Red, Ly6G-V450, CD44-V450, TCRβ-APC, CD11b-APCCy7, CD3-APC (all BD Biosciences); 7/4-PE (AbD Serotec), CD8-PECy7 (Invitrogen), F4/80-APC (Invitrogen). Samples were then washed before analysis on a CyAn™ ADP Flow cytometer using Summit software (Beckman Coulter). Cell subsets were defined as the following: eosinophils (F4/80^intCD11b^intSSC^hi), 7/4⁻ monocytes (F4/80^lowCD11b^intSSC^low7/4⁻), 7/4⁺ monocytes (F4/80^lowCD11b^intSSC^low7/4⁺), myeloid dendritic cells (DCs) (CD11b⁺CD11c⁺MHCII⁺), CD4⁺ T cells (CD3⁺αβTCR⁺CD4⁺), CD8⁺ T cells (CD3⁺αβTCR⁺CD8⁺), NK (NK1.1⁺αβTCR⁻CD3⁻) and NKT (NK1.1⁺αβTCR⁺CD3⁺) cells.