Cell staining was performed in the presence of purified rat anti-mouse CD16/32 antibody (BD). Antibodies used for staining were as follows; anti-Ly6G-PE (1A8, BD), anti-CD11b-APC (M1/70, eBioscience), anti-Ly6G-APC-Cy7 (1A8, BD), anti-Ly6C-APC-Cy7 (AL-21, BD), mouse biotin-conjugated anti-CD45 (30-F11, eBioscience), anti-streptavidin-V500 (BD), and anti-7-AAD (BioLegend).
For intracellular staining for phosphorylated p38 MAPK, 1 × 106/ml of bone marrow-derived neutrophils were stimulated with 1 μg/ml of R848 (Enzo Life Sciences) for 30 min in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS), 100 μg/ml of L-glutamine (Sigma-Aldrich), 100 U/ml of penicillin (Sigma-Aldrich), 100 μg/ml of streptomycin (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Stimulated neutrophils were stained with anti-CD11b-APC (M1/70, eBioscience) and anti-Ly6G-APC-Cy7 (1A8, BD) antibodies in the presence of purified rat anti-mouse CD16/32 antibody (BD), fixed with Lyse/Fix Buffer (BD), and permeabilized by BD Phosflow Perm Buffer II (BD). Then, neutrophils were intracellularly stained with Alexa Fluor 488 Mouse IgG1κ isotype control (BD) or Alexa Fluor 488 mouse anti-p38 MAPK (pT180/pY182) (BD).
Flow cytometric analysis was performed using MoFlo XDP and data were analyzed using FlowJo Software (Tree star).
For intracellular staining for phosphorylated p38 MAPK, 1 × 106/ml of bone marrow-derived neutrophils were stimulated with 1 μg/ml of R848 (Enzo Life Sciences) for 30 min in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS), 100 μg/ml of L-glutamine (Sigma-Aldrich), 100 U/ml of penicillin (Sigma-Aldrich), 100 μg/ml of streptomycin (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Stimulated neutrophils were stained with anti-CD11b-APC (M1/70, eBioscience) and anti-Ly6G-APC-Cy7 (1A8, BD) antibodies in the presence of purified rat anti-mouse CD16/32 antibody (BD), fixed with Lyse/Fix Buffer (BD), and permeabilized by BD Phosflow Perm Buffer II (BD). Then, neutrophils were intracellularly stained with Alexa Fluor 488 Mouse IgG1κ isotype control (BD) or Alexa Fluor 488 mouse anti-p38 MAPK (pT180/pY182) (BD).
Flow cytometric analysis was performed using MoFlo XDP and data were analyzed using FlowJo Software (Tree star).