Anti f4 80 af488

Cells were prepared from the aorta of aging mice for multicolor flow cytometric analysis of M1 and M2 macrophages in aorta after elimination of red blood cells (RBC) with RBC lysis buffer using LSR Fortessa™ X-20 (BD Bioscience, CA, USA). Cells in aorta were obtained and prepared using enzyme digestion for flow cytometry analysis for macrophages and M1 and M2 as described [18 18 . Gjurich, B.N. • Taghavie-Moghadam, P.L. • Galkina, E.V. ]. Living cells were gated using yellow LIVE/DEAD™ Fixable Dead Cell Stain Kits (1:1,000, Invitrogen, Carlsbad, CA, US). CD45-positive cells were identified as leukocytes. F4/80-positive cells were defined as monocytes/macrophages in the aorta. M1 macrophages were identified as F4/80 + /CD80 + /CD206 -cells, and M2 as F4/80 + /CD80 -/CD206 + cells. Anti-F4/80 AF488 (#123120, dilution factor of 1:125), and anti-CD206 PE (#141706, dilution factor of 1:125) were from Biolegend (San Diego, CA, U.S). Anti-CD80 BV786 (#740888, at 1:125) was from BD Pharmingen (San Jose, CA, US). For analysis of GFP-positive macrophages (see below), anti-F4/80 AF594 (#123140, dilution factor of 1:125) was used since Anti-F4/80 AF488 shares the same color as GFP. Data analysis was performed using FlowJo software.