Apc anti human mouse trem2

For flow cytometric analysis, heart tissues were digested with collagenase type II solution (Worthington Biochemical Corporation, USA) at a concentration of 500 unit/ml for 40 min at 37 °C and homogenized in gentleMACS Dissociator (Miltenyi Biotec, USA). After homogenization, the tissues were passed through a 40 μm cell strainer, washed with HBSS Buffer, and resuspended in FACS staining buffer. For FACS analysis, the following antibodies were used: FITC anti-mouse CD45 antibody (1 μg/mL; BD; #553080), BV421 anti-mouse Ccr2 (1 μg/mL; BioLegend; #150605), APC anti-human/mouse Trem2 (1 μg/mL; R&D system; #FAB17291A), and PE-Cy7 anti-human/mouse Arg-1 (1 μg/mL; Invitrogen; #25-3697-82). CD45 positive cells were sorted by flow cytometry sorter (Beckman coulter, USA). After staining each sample, FACS analysis was performed using the FACS Canto II (BD) and Flow-jo software (TreeStar Inc., USA).

For FACS or flow experiments, single-cell suspensions of cells were resuspended in a 2% FBS solution and then stained with appropriate antibodies at a 1:100 dilution for 60 min at 4 °C in the dark. Antibodies used were APC anti-human/mouse TREM2 (R&D; FAB17291A), APC/Cy7 anti-mouse CD45 (BioLegend; 103116), Alexa700 anti-mouse CD45.2 (BioLegend; 109822), PerCP/Cy5 anti-mouse CD11b (BioLegend; 101228), BV605 anti-mouse Cd11c (BioLegend; 117334), FITC anti-mouse Ly6d (BioLegend; 138606), PE/Cy7 anti-mouse Vcam1 (BioLegend; 105720), BC510 anti-mouse F4/80 (BioLegend; 123135), PE/Cy7 anti-human CD326 (BioLegend; 324222), and PE anti-human VCAM1 (BioLegend; 305805). Before sorting or flow, SytoxBlue (Thermo Fisher; S34857) was used for a viability dye at a 1:1000 dilution. FACS experiments were run on FACSAria II, and flow experiments were run on LSRII instruments, where BD FACSDiva v8.0.1 was used for data collection. Subsequent analysis was done in FlowJo v10.6.