Efluor450 anti cd11b m1 70

We used flow cytometry to identify neutrophils (Ly6G⁺; CD11b⁺), monocytes (Ly6G^-; CD11b⁺), and eosinophils (SSC^high; Siglec-F⁺), in air pouch exudates. Red blood cells were lysed by treatment with pH 7.3 ACK lysis buffer (0.15 M NH₄Cl, 1 mM KHCO₃, and 0.1 mM EDTA, pH 8.0) 2 times for 5 min each. Cells were blocked with unconjugated anti—CD16/CD32 antibodies (BioXcell) on ice for 5 min and then stained with APC/Cy7 anti-Ly6G (1A8, BD Biosciences), PE anti-Siglec-F (E50-2440, BD Biosciences) and eFluor450 anti-CD11b (M1/70, eBioscience) antibodies on ice for 30 min. Data were acquired using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo 9.5.3. software (TreeStar). Dead cells (identified by staining with LIVE/DEAD aqua amine; Invitrogen) were not included in the analysis.

BM-derived mφs (BMDMs) were trypsinized and resuspended in flow cytometry buffer (PBS with 2% FBS). Following this, 1 × 10⁶ cells were blocked with 0.5 μg rat anti-mouse CD16/32 (2.4G2; BD Pharmingen) in a volume of 100 μl for 30 min on ice. Cells were then stained with the following Abs: 5 μg/ml eFluor450 anti-F4/80 (BM8; eBioscience), 1.25 μg/ml PE Cy7 anti-CD115 (25-1152; eBioscience), or 1.2 μg/ml eFluor450 anti-CD11b (M1/70; eBioscience). Flow cytometry was performed with a Fortessa LSR II (BD Biosciences) and analyzed using FlowJo software.