Cardiac Inflammatory Cell Profiling after MI

From each treatment group, mice not subjected to functional and histological analysis were used for flow cytometric analysis of cardiac inflammatory cell invasion. Mice were sacrificed on days 1, 3, and 7 after MI. Each time point, 3 hearts were harvested per group. Sham animals were used as controls to determine base line characteristics. Total cardiac cell numbers were determined with a Sysmex cell counter (Sysmex America, Inc. Mundelein, Illinois, US). Single-cell suspensions were stained for flow cytometry with primary antibodies before analysis using a FACSCanto II (BD Biosciences, San Diego, CA, USA). The following antibodies were used: anti–CD90-APC, 53–2.1,–B220-APC, RA3-6B2,–CD49b-APC, DX5,–NK1.1-APC, PK136,–Ly-6G-APC, 1A8, CD11b-eFluor 450, M1/70,–CD11c-FITC, HL3,–I-A^b -FITC, AF6-120.1,–Ly-6C-PE, AL-21,–CD11c-PE, HL3 (All above antibodies are from BD Biosciences),–F4/80-FITC, C1:A3-1 (ABD Serotec, Kidlington, UK). Monocytes were identified as CD11b high (CD90/B220/CD49b/NK1.1/Ly-6G) low (F4/80/I-A^b /CD11c) low Ly-6C high/low as previously described [26 (link),28 (link)]. Macrophages were identified as CD11b high, F4/80 high. Dendritic cells were identified as CD11b, I-A^bandCD11c high. Neutrophils were identified as CD11b, Ly-6G high. The analysis of the acquired data was done with FlowJo software version 7.6.1 (Tree Star Inc. Ashland, OR, USA).