Cd45.1 efluor 450

Blood was washed in PBS, stained with live/dead fixable blue dead cell staining kit (Invitrogen), washed in PBS and blocked in 2% FBS-PBS / 0.02% sodium azide plus Fc-block (Anti-CD16/32 antibody 1:200, BD Biosciences). Surface antigens were detected by incubation for 30 min at 4°C with conjugated antibodies including CD45.1-eFluor 450 (eBioscience Cat# 48-0453-82, RRID:AB_1272189), CD45.2-FITC (BD Biosciences Cat# 553772, RRID:AB_395041), CD3e-APC (BD Biosciences Cat# 561826, RRID:AB_10896663), CD19-APC-H7 (BD Biosciences Cat# 560143, RRID:AB_1645234), CD11b-PE (BD Biosciences Cat# 562287, RRID:AB_11154216) and GR1-Alexa Fluor 700 (BioLegend Cat# 108422, RRID:AB_2137487). Following washing with 2% FBS-PBS 0.02% sodium azide, red cells were lysed and leukocytes fixed by incubating in lyse/fix solution (BD Biosciences). Cells were washed with PBS and analyzed on an LSR-II cytometer (Becton Dickenson). Data were processed using FlowJo Software (Tree Star).

Single-cell suspensions of BM and skin-infiltrating leukocytes were incubated at 4 °C for 20 min in staining buffer (1 × PBS with 0.1% bovine serum albumin and 0.1% sodium azide) containing the appropriate antibody mix. NOS2-AF488, CD45.1-eFluor 450, Ly6G-FITC, Ly6G-PE, Ly6G-PE.Cy7, CD11b-PE, CD11b-APC, F4/80-PE, CD115-APC, Ly6C-APC, CD45-PE, B220-PE, CD4-APC, CD8-APC, and CD105-APC antibodies were obtained from eBioscience. Lineage cocktail-APC, CD11c-APC.Cy7, α-BrdU-FITC and CD34-PE antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Arginase-FITC antibody was purchased from R&D systems (Minneapolis, MN, USA). For the flow cytometric analysis of Lineage-positive cells, the cells were stained with biotinylated Lineage cocktail obtained from BD Pharmingen, then labeled with streptavidin-V450 (BD Pharmingen). Data were collected using a FACSCalibur or LSRII-Green (both BD Pharmingen) and were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).