Rat anti mouse cd16 cd32

Cells were blocked with rat anti-mouse CD16/CD32 (BioLegend) and stained with: LIVE/DEAD™ Fixable Aqua Dead Cell (ThermoFischer), PerCpCy5.5 Rat anti-mouse IgD (BioLegend), Pacific Blue Rat anti-mouse B220 (BioLegend), PE Cy7 Hamster anti-mouse CD95 (BD Biosciences), PE Rat anti-mouse T- and B-cell activation antigen (BD Biosciences). Samples were attained on BD LSRFortessa X20 and analyzed using FlowJo software.

Total cell suspensions were prepared from lymph nodes, thymus, spleen and bone marrows, and red blood cells were lysed by incubation with ACK lysis. Single cell suspensions were pre-incubated with purified rat anti-mouse CD16/CD32 (Biolegend, 101302) to block FcγR, and then stained with fluorochrome conjugated cell surface antibodies: CD45 (BD Bioscience, 553081), CD11b (Biolegend, 101212), Gr-1 (Biolegend, 108406), CD3 (Biolegend, 100221), B220 (Biolegend, 103212), CD4 (Biolegend, 100540), and CD8 (Biolegend, 100706). Data from immunofluorescent staining of cells were acquired on Guava easyCyte HT (Millipore) and analyzed using guavasoft 3.1.1.1. The gating strategy for FCAS is shown in Supplementary Fig. 9.

Single-cell suspensions were stained and analysed using a FACS LSRFortessa™ system (BD Biosciences). Dead cells were excluded with the LIVE/DEAD Fixable Violet Dead Cell staining kit (Life Technologies). The cells were then incubated for 10 minutes with purified rat anti-mouse CD16/CD32 (Biolegend, clone 93) and normal mouse serum (Interchim) before being stained with various monoclonal antibodies for 20 minutes in the dark on ice. For mouse cells, lineage-positive cells were excluded using the PerCP5.5-conjugated anti-CD3 (17A2), anti-NK1.1 (PK136), anti-CD19 (6D5), anti-Ly6G (1A8) (Biolegend). PerCP-conjugated anti-CCR3 (83103) added to the lineage staining to exclude eosinophils was from R&D. Alexa Fluor 700-conjugated anti-Ly6C (AL21) was from BD Pharmingen. PECF594-conjugated anti-CD11c (HL3) was from BD Horizon. Allophycocyanin-Cy7-conjugated anti-CD11b (M1/70), PE-Cy7 anti-CD8 (53-6.7), Brilliant violet 510-conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2), Brilliant violet 650-conjugated anti-CD45.2 (104), were all from Biolegend. PE-conjugated anti-CCR2 (475301) was from R&D systems. The data were analysed with Flowjo software V10.1 (TreeStar).