Anti cd64 pe

Cell suspensions were Fc-Receptor blocked and stained with the following antibodies (all from Biolegend unless indicated otherwise): (Myeloid panel) anti-Ly6G-FITC (1A8; 1.25 μg/ml), anti-CD64-PE (X54-5/7.1; 2 μg/ml), anti-CD11c-PerCP-Cy5.5 (N418; 1 μg/ml), anti-CD11b-PE-Cy7 (M1/70; 0.2 μg/ml), anti-Ly6C-AlexaFluor700 (HK1.4; 2 μg/ml), anti-MHCII-APC-Cy7 (M5/114.15.2; 0.067 μg/ml), anti-TREM-1-eFluor660 (TR3MBL1 from eBioscience; 0.2 μg/ml), anti-CD45-BrilliantViolett (30-F11; 1 μg/ml). (Lymphocyte panel) anti-CD8b-FITC (53-5.8; 0.8 μg/ml), anti-TCRab-PE, anti-GL7-PerCP-Cy5.5 (GL7; 1 μg/ml), anti-CD19-PE-Cy7 (6D5; 0.5 μg/ml), anti-CD11b-PacificBlue (M1/70; 0.25 μg/ml), anti-NK1.1-AlexaFluor700 (PK136; 2.5 μg/ml), anti-CD4-APC-Cy7 (RM4-5; 0.25 μg/ml), anti-BTLA-AlexaFluor647 (6A6; 2.5 μg/ml), anti-CD45-BrilliantViolett (30-F11; 1 μg/ml). As an isotype control for the anti-TREM-1-e-Fluor660 a mouse anti rat IgG2a-eFluor660 antibody (eBR2a; 0.2 μg/ml) from eBioscience was used. Dead cells were excluded using DAPI (Invitrogen) at a final concentration of 0.5 μg/ml. All samples were acquired on a LSRII SORP (BD Biosciences, San Diego, CA) and analyzed using the FlowJo Software (Tree Star, Ashland, OR).

Binding to FcRs (CD16/32) was blocked by incubating the cells on ice for 30 min with 2.4G2 hybridoma supernatant, except in experiments where the FcRs were stained when the blocking was performed by incubating the cells in 5% BSA in FACS wash solution (0.5% BSA, 2 mM NaN₃, 2 mM EDTA in PBS). After blocking, the antibodies were added for 1 h: anti-Sn-biotin (3D6 and SER-4 mAbs produced in-house); anti-TIM-4-PE, anti-CD16/32-PE, anti-CD64-PE (all eBioscience); anti-FcγRIIB Ly17.2 (kind gift from Prof. J. Ravetch, The Rockefeller University, NY, USA) that was directly conjugated to Alexa 488 fluorescent dye using an antibody conjugation kit (Invitrogen). Corresponding isotypes were used at the same concentration in each staining. After incubating the cells with antibodies, the cells were washed in FACS buffer and, if necessary, incubated with streptavidin-APC (BD Bioscience, San Jose, CA, USA) for a further 30 min on ice and washed again. Cells were fixed with 1% formaldehyde in PBS and data were acquired with FACSCalibur (BD Bioscience). Analysis was performed using FlowJo 7.6.4 (Tree Star).