Percp cy5.5 anti mouse cd4

Twenty four weeks post final DNA immunization, the splenocytes were isolated from the immunized mice (n = 6 per group). CD4⁺ memory cells were stained with PerCP-Cy5.5-anti-mouse CD4 (Biolegend), FITC-anti-mouse CD44 (Biolegend), PE-anti-mouse CD62L (Biolegend), and APC-Cy7-anti-mouse CD25 (Biolegend). CD8⁺ memory cells were stained with PerCP-anti-mouse CD8 (Biolegend), PE-anti-mouse CD62L (Biolegend), and Pacific blue-anti-mouse CD45R (Biolegend) antibodies. The stained cells were fixed with Cytofix/Cytoperm Buffer™ (Becton Dickson) and then analyzed with a FACS Canto II flow cytometer with FACSDiva software.

Whole lungs were dissociated into single‐cell suspensions using the gentleMACS Dissociator. Red blood cell lysing buffer (Sigma‐Aldrich) was used for red cell lysis.
Lung cells were blocked with anti‐mouse CD16/32 (101319; Biolegend) and then stained with antibodies PerCP anti‐mouse/human CD11b (101229; Biolegend), Brilliant Violet 421 anti‐mouse F4/80 (123137; Biolegend), APC/Cy7 anti‐mouse CD45 (103116; Biolegend), PerCP/Cy5.5 anti‐mouse CD11c (117328; Biolegend), PE Siglec‐F (552126; BD Biosciences), PerCP/Cy5.5 anti‐mouse CD4 (100540; Biolegend), PE/Cy7 anti‐mouse CD3ε (100320; Biolegend), Brilliant Violet 421 anti‐mouse CD335 (NKp46) (137612; Biolegend), APC anti‐mouse CD8a (100712; Biolegend), Brilliant Violet 421 anti‐mouse Ly‐6G/Ly‐6C (Gr1) (108433; Biolegend) and Zombie Aqua Fixable Viability Kit (423102; Biolegend). Flow cytometric data acquisition was performed on BD FACS Canto II machine and data analysis was performed using FlowJo software.
Gating for CD45+AquaZombie−Siglec‐F+CD11c+Gr1− cells was used for AMs; CD45+AquaZombie−F4/80+CD11b+Gr1− for IMs CD45+AquaZombie−NKp46+ for natural killer (NK) cells, CD45+AquaZombie−CD3+CD8+ for CD8 T cells.

Aortic cell suspension and SVC were resuspended in 2% Fc Block (553142, BD Pharmingen) and blocked for 30 min. Then fluorophore-conjugated primary antibodies were incubated for 30 min: PE/Cy7 anti-mouse CD45 (102114, Biolegend), APC anti-mouse F4/80 (MCA497APC, Biorad), PE anti-mouse CD11b (RM2804, Invitrogen), PE-Texas Red anti-mouse CD11c (MCD11C17, Invitrogen), BV421 anti-mouse IAIE (107631, Biolegend), BUV395 anti-mouse B220 (56793, BD Biosciences), BV786 anti-mouse CD3 (564379, BD Biosciences), PerCP/Cy5.5 anti-mouse CD4 (100540, Biolegend), APC-Cy7 anti-mouse CD8 (557654, BD Biosciences), and BV510 anti-mouse CD25 (740106, BD Biosciences) in FACS buffer (1X HBSS (Thermo Fisher Scientific), 2% BSA and 0.5 mM EDTA). All antibodies were been used at 1:200. After one wash in FACS buffer, cells were resuspended in FACS buffer containing 1 µg/mL DAPI (Invitrogen). Immune cells profiling was performed using a FACSAria II cell sorter (BD Biosciences) and data were analyzed with FlowJo v10. Adipose tissue macrophages (FB and FBC) were sorted and collected.