Lsr 2 flow cytometer unit

Flow cytometry analysis was performed by the standard method, as previously described [15 (link),16 (link)] via an LSR II flow cytometer unit (BD Biosciences, San Jose, CA, USA). Briefly, mice were euthanized and perfused with 10 mL PBS via left ventricle to remove circulating immune cells; then heart tissue was isolated and minced into small pieces, followed by digestion with HBSS with Collagenase IV (2 mg/mL, #LS004188, Worthington Biochemical Co., Lakewood, NJ, USA), Dispase II (1.2 U/mL, #D4693, Sigma) and 0.9 mM CaCl₂, then incubated at 37 °C for 45 min with gentle agitation. Next, tissue was filtered through 40 µm cell strainer and centrifuged at 500× g at 4 °C for 5 min. The pellet was re-suspended in flow cytometry soring buffer (HBSS with 1 mM EDTA, 25 mM HEPES and 1% FBS). After blocking with CD16/32 antibody, BMDMs and macrophages collected from mouse hearts (above) were surface-stained with antibodies against CD11b (47-0112-80, eBioScience, San Diego, CA, USA), CD45.2 (109816, Bio-Legend, San Diego, CA, USA), CD206 (MCA2235 A700, Bio-Rad, Hercules, CA, USA), F4/80 (123135, BioLegend), Ly6C (17-5932-82, eBioScience), Ly6G (127628, Bio-Legend), MHC-II (46-5321-82, eBioScience). The data were analyzed using FCSexpress software (ACEA Biosciences, San Diego, CA, USA).

Flowcytometric analysis was performed by standard methods with an LSR II flow cytometer unit (BD biosciences). Cells were digested and immunostaining was performed with Annexin V and PI apoptosis detection kits (BD Biosciences). After staining, the cells were filtered through a cell strainer and 100,000–1,000,000 events were acquired for each sample. Flow cytometry data were analyzed with BD FACSDiva software.