Cd206 alexa fluor 647

Cells were detached and washed once with 1x PBS and resuspended in FACs buffer (0.5% BSA, 2 mM EDTA in PBS). Staining with extracellular fluorochrome coupled-antibodies was carried out in 100uL of FACs buffer for 20 minutes in the dark at 4 °C. Antibodies used for macrophage phenotyping were: CD80 (BV605 BD Biosciences), CD83 (BV650, BD Biosciences), CD86 (BV786, BD Biosciences), CD200R (PE, Biolegend), CD206 (Alexa-Fluor647, BD Biosciences), CD209 (BV421, BD Biosciences). Viability staining was performed by adding one drop of Sytox green flow reagent (ThermoFisher Scientific) to samples prior to analysis. Compensation was performed using either Ultracomp eBeads plus compensation beads (Invitrogen). All samples were rewashed in FACs buffer once before being analyzed using a BD LSR-Fortessa X20 cell analyzer. Data were analyzed using the FlowJo software (BD Biosciences).

Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells were then incubated with an antibody at 4 °C for 30 min in the dark in PBS with 2% normal FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to standard techniques. To characterize splenic cells, the monoclonal antibodies used spleen cell suspensions prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells’ suspensions were incubated for 20 min with the following antibodies. All antibodies were obtained from Biolegend (San Diego, CA, USA) and used at a dilution of 1:100 unless otherwise mentioned: CD3 FITC, CD4 APC Fire 750 (BD Biosciences, San Jose, CA, USA), CD8 BV 605, CD69 PEDazzle594, CD40 PercPCy5.5, B220 APC, CD44 BV650, MHC II eFluor450 (DAPI) (eBioscience, San Diego, CA, USA), F4/80 BV711 (dilution 1:200), CD11b PercP Cy5.5, CD80 BV421, CD86 FITC, CD206 Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA. Dilution 1:200), Ly6C PECy7.