Anti cd4 percp clone rm4 5

Cells were counted and Fc receptors were blocked using anti‐CD16/CD32 (clone 2.4G2) (catalog no. 553141) from Becton‐Dickinson (Franklin Lakes, NJ, USA) to prevent non‐specific binding. Cells for immunofluorescence staining were labelled with monoclonal antibodies: anti‐CD3e FITC (clone 145‐2C11) (catalog no. 11‐0031‐86) or Armenian hamster IgG isotype control FITC (clone eBio299Arm) (catalog no. 11‐4888‐81), anti‐CD11c APC (clone N418) (catalog no. 17‐0114‐82) or Armenian hamster IgG isotype control APC (clone eBio299Arm) (catalog no. 17‐4888‐82), and anti‐B220 PE (clone HIS24) (catalog no. 12‐0460‐82) or mouse IgG2b kappa isotype control PE (clone eBMG2b) (catalog no. 12‐4732‐82) from eBioscience (Carlsbad, CA, USA) and anti‐CD4 PerCP (clone RM4‐5) (catalog no. 553052) and rat IgG2a, κ isotype control PerCP (clone R35‐95) (catalog no. 553933) from BD Pharmingen (San Diego, CA, USA) at 4 °C for 30 min. Excessive antibodies were washed off and labeled cells were analyzed using a flow cytometer (BD ACCURI C6). Data were analyzed usinf flowjo (Tree Star Inc., Ashland, OR, USA) and prism (GraphPad Software Inc., San Diego, CA, USA).

The following Abs were used for cell surface staining: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Texas Red (clone 53.6.7), anti-CD44 PE-Cy7 (clone IM7), anti-B220 PE Texas Red (clone RA3-6B2), anti-NK1.1 FITC (clone PK136), anti-CD69 Alexa Fluor 647 (clone H1-2F3), anti-Ly6C APC-Cy7 (AL-21), anti-Ly6G PE-Cy7 (clone 1A8) (Biolegend, San Diego, CA, USA), anti-CD4 APC e-Fluor 780 (clone GK1.5), anti-CD62L PE-Cy5 (clone MEL-14) (eBiosciences, San Diego, CA, USA), anti-CD45RB PE (clone 16A), anti-CD3 Alexa Fluor 647 (clone 17A2), anti-CD4 PerCP (clone RM4-5), anti-CD8 PE (clone 53-6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-CD45 PE (clone 30-F11), anti-CD11c FITC (clone HL3) and anti-CD11b Alexa Fluor 700 (clone M1/70) (BD Biosciences, San José, CA, USA). The abs for intracellular staining included anti-FoxP3 Alexa Fluor 488 (clone MF23), anti-IFNγ Alexa Fluor 700 (clone XMG1.2), TNFα PE-Cy7 (clone MP6-XT22), IL-2 FITC (clone JES6-5H4) (BD Biosciences) and anti-CTLA-4 PE (clone UC10-4F10-11) (Biolegend). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Molecular Probes, Eugene, OR) was used for dead cell exclusion.