For surface staining, cells were washed with PBS and blocked with PBS plus 5% FBS, 2.5% mouse serum and 0.5% anti Fcγ III/II (anti-CD16/32) for 15min. Cells were washed with PBS and incubated with viability dye (1:500, eBioscience) and antibodies (1:200) for 15 min. Cells were washed with PBS and analyzed by LSR II (BD Biosciences). Flow cytometric data were further analyzed with FlowJo software (TreeStar).
For intracellular staining, cells were incubated with Brefeldin A (1:1000, eBioscience) in RPMI media plus 10% FBS in 37 C for 4–5h. Then cells were washed, blocked and stained according to the Intracellular Staining protocol (eBioscience). Cells were washed with PBS and analyzed by BD LSR II.
Antibodies used in this paper include Ly6C-APC Cy7, Ly6C-APC, F4/80-PE, F4/80-e450, Ly6G 1A8-PE Cy7, Ly6G 1A8-FITC (BD), CD45.2-APC, CD45.2-AF700, CD45.1-PE, CD45.1-AF700, CD3-e450, NK1.1-AF780, CD11b-PerCP Cy5.5 (BD), CD206-PE, Arginase1-PE (R & D), Anti-mouse Relm-α antibody (PeproTech). Antibodies were from eBioscience unless stated otherwise.
For intracellular staining, cells were incubated with Brefeldin A (1:1000, eBioscience) in RPMI media plus 10% FBS in 37 C for 4–5h. Then cells were washed, blocked and stained according to the Intracellular Staining protocol (eBioscience). Cells were washed with PBS and analyzed by BD LSR II.
Antibodies used in this paper include Ly6C-APC Cy7, Ly6C-APC, F4/80-PE, F4/80-e450, Ly6G 1A8-PE Cy7, Ly6G 1A8-FITC (BD), CD45.2-APC, CD45.2-AF700, CD45.1-PE, CD45.1-AF700, CD3-e450, NK1.1-AF780, CD11b-PerCP Cy5.5 (BD), CD206-PE, Arginase1-PE (R & D), Anti-mouse Relm-α antibody (PeproTech). Antibodies were from eBioscience unless stated otherwise.