Anti cd11b pe cy7 clone m1 70

Protamine was obtained from Valeant Pharmaceuticals Germany GmbH (Germany). Following antibodies were purchased from eBioscience (Germany): anti-NK1.1-PerCP-Cy5.5 (clone PK136), anti-CD4-APC (clone RM4-5), anti-CD11c-APC (clone N418), anti-CD8-PE-Cy7 (clone 53-6.7) and anti-F4/80-APC-eFluor780 (clone BM8). Anti-CD11b-PE-Cy7 (clone M1/70), anti-CD3-APC-Cy7 (clone 145-2C11) and anti-CD45.2-HV450 (clone 104) were obtained from BD Bioscience (Germany). SIINFEKL and control peptides were purchased from Bachem AG (Germany). Collagenase type IV was obtained from Sigma-Aldrich (Germany) and DNAse I from Roche (Germany).

Single cell suspensions of footpad cells were filtered over a 70-µm nylon filter mesh and incubated in saturating doses of purified anti-mouse Fc receptor (CD16/32, clone 2.4G2, BD Bioscience) in 1 ml PBS + 5% FCS for 10 min on ice to prevent antibody binding to Fc receptor. The isolated cells were stained on ice with various fluorescent monoclonal antibodies (mAbs) combinations in PBS + 5% FCS for 30 min. Anti-Ly6G-FITC (clone 1A8, BioLegend), anti-CD18-PE (clone M18/2, eBioscience), anti-CD11b-PECy7 (clone M1/70, BD Biosciences), anti-Ly6C-APC (clone AL-21, BD Biosciences), anti-CD62L-APCCy7 (clone MEL14, eBioscience), anti-MHCII-BV421 (clone M5/114.15.2, BD Biosciences), and anti-CD45-BV510 (clone 30-F11, BD Biosciences). A live/dead near-IR stain (ThermoFischer Scientific) was performed in PBS only. Unbound mAbs were discarded by washing the samples with PBS + 5% FCS and centrifugation (1,400 rpm, 7 min, 4°C). Analyses were performed using a FACS Canto II flow cytometer (BD Biosciences) and data were processed using FlowJo software (Tree Star Inc.). Cells were gated according to size and scatter to eliminate debris from analysis.

Cells counts were performed using a Z2 Coulter Counter (Beckman Coulter, USA). 3X10⁶ cells from single cell suspensions were incubated at 4 °C for 20 min with the following fluorochrome-conjugated anti-mouse markers: anti-CD45 APC-Cy7 (clone 30-F11; Biolegend, San Diego, USA), anti-CD11b PE-Cy7 (clone M1/70; BD Biosciences), anti-CD11c Pacific Blue (clone N418; Biolegend), anti-Ly6C FITC (clone HK1.4; Biolegend), anti-Ly6G Alexa Fluor 647 (clone 1A8; Biolegend), anti-F4/80 APC (clone BM8; eBioscience), and anti-CD206 (mannose receptor; MR) Alexa Fluor 488 (clone C068C2; Biolegend). Fc receptor block (anti-CD16/32 antibody) was added to all markers cocktails. Intracellular CD206 labelling was performed using a CytoFix/CytoPerm kit (BD Biosciences, USA). After surface receptor labelling, cells were permeabilized and incubated with the marker for 30 min at 4 °C in the dark before being washed twice in 1× Perm/Wash buffer (BD Biosciences) and resuspend in FACS buffer. A BD FACS Canto II flow cytometer (BD Biosciences) was used to acquire data. Data was analysed using FlowLogic FCS analysis software (Inivai Technologies, Melbourne, Australia).