Apc conjugated anti f4 80

The cells were washed twice and cells (1 × 10⁶) were suspended in 50 μL PBS supplemented with 1% FBS and stained for 20 min at 4 °C with directly conjugated fluorescent antibodies (1:500). Antibodies were as follows: eFluor450-conjugated anti-CD11b (eBioscience, Santa Clara, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-Gr-1 (BD Biosciences, Franklin Lakes, NJ, USA), allophycocyanin (APC)-conjugated anti-Gr-1 (eBioscience), FITC-conjugated anti-CD11c (eBiosciences), APC-conjugated anti-F4/80 (eBioscience), isotype control Rat IgG2a, and isotype control Rat IgG2b (BD Biosciences). Stained cells were analyzed with a FACS Canto flow cytometer using FACS Diva software (BD Biosciences), and the data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA). Cells were sorted with a FACS Aria flow cytometer (BD Biosciences), washed twice, and suspended in PBS. Cells (2 × 10⁵) were resuspended in 100 μL PBS and centrifuged onto microscopic slides using a Cytospin-4 (Thermo Scientific, Waltham, MA, USA). Slides were then stained by May-Grunwald Giemsa according to a standard protocol.

The following antibodies were purchased from BD Biosciences (San Jose, CA, USA) and used for flow cytometry analysis: FITC-conjugated anti-Ly6C, anti-CD69, anti-CD11b, and anti–TNF-α; PE-conjugated anti-Ly6G, anti-CD8, and anti–IFN-γ; PerCP-Cy5.5–conjugated anti-CD3; PE-Cy7–conjugated anti-NK1.1 and anti-CD45; allophycocyanin(APC)-conjugated anti-CD45.2, and anti-CD11c; APC-Cy7–conjugated anti-CD4 and anti-CD45.1. APC-conjugated anti-F4/80 were purchased from eBioscience (San Diego, CA, USA). Stained cells were collected using a BD LSR II (BD Biosciences) flow cytometer, and data were analyzed using FlowJo 7.6 software (Tree Star, Ashland, OR, USA). For NK cell depletion, mice were injected with 30 μl anti-ASGM1 antibody once a week (Wako Co., Tokyo, Japan). For NK1.1⁺cell depletion, mice were intraperitoneally injected with 200 μg anti-NK1.1 (Clone PK136, purified from ascites).

At 3 h post-study, a subset of animals (n = 6 naïve, n = 5 sham-subjected to vessel cannulation and terminal anesthesia only, n = 5 THS) were intracardially perfused with heparinized saline under terminal anesthesia. The hearts were then immediately isolated, cut into 1 mm³ pieces and dissociated by incubation with papain (Merck, UK) and DNAse I (ThermoFisher Scientific, UK) for 30 min at 37°C. Following lysis of residual red blood cells (RBC Lysis Buffer, Biolegend, UK), cell suspensions were incubated with CD16/CD32-block (Biolegend, UK) for 30 min at 4°C, followed by incubation for 30 min at 4°C with PE-conjugated anti-CD45 to define immune cell populations, and FITC-conjugated anti-Ly6C/G (clone RB6-8C5) and APC-conjugated anti-F4/80 (all ThermoFisher Scientific, UK) to differentiate neutrophils (F4/80Neg, Ly6C/GHi) from pro-inflammatory (F4/80Pos, Ly6C/GInt) and anti-inflammatory (F4/80Pos, Ly6C/GLow) monocytes/macrophages (19 (link), 20 (link)). Cells were then analyzed by flow cytometry using a BD FACSCanto II instrument (BD Biosciences) and FlowJo 8.8.1 software (TreeStar Inc., FL, USA). In all cases, 20,000 singlet CD45Pos events were analyzed per sample; positive staining was defined by inclusion of fluorescence-minus-one controls for all antigens.