Efluor 450 anti f4 80 bm8

The mouse lungs were cut into small pieces and incubated in Iscove's Modified Dulbecco's Medium (Invitrogen) containing 1mg/ml collagenase D (Roche, Mannheim, Germany) and 20 U/ml DNase I (Roche) at 37°C with shaking for 1–2 hours. After treating with Ammonium-Chloride-Potassium lysing buffer and filtering by a 70 µm nylon mesh, 1×10⁶ cells were stained with a combination of monoclonal antibodies (mAbs) of PE-Cy5-anti-CD11b (M1/70, eBioscience), eFluor 450-anti-F4/80 (BM8, eBioscience), and Alexa Fluor 700-anti-Ly-6G (RB6-8C5, eBioscience); or a combination of mAbs of eFluor 450-anti-CD3 (17A2, eBioscience), FITC-anti-NK1.1 (PK136, BioLegend, San Diego, CA, USA), PE-anti-CD4 (RM4-5, BD), and APC-anti-CD8a (53-6.7, BD); or their relevant isotype-specific antibodies as described previously [26] (link), [28] (link). Before running samples, counting beads were added into the stained cells (Molecular Probes, Carlsbad, CA, USA). All samples were acquired on LSR II and analyzed by FlowJo software (TreeStar, Ashland, OR, USA).

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.