Pe cy5 anti cd11b m1 70

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).

Control rat IgG (2A3) and anti-PD-1 (RMP1-14) used in vivo were obtained from BioXcell. Dosing per injection was 250 μg of 2A3 and 250 μg of RMP1-14.
Staining antibody included FITC anti-CD45.2 (104), PE-Cy7 anti-CD4 (RM4-5), PerCP-Cy5.5 anti-CD8a (53-6.7), BV421 anti-PD-1 (J43) from BD Biosciences; APC anti-CD25 (PC61.5), PE anti-Foxp3 (FJK-16s), and PE-Cy5 anti-CD11b (M1/70) from eBiosciences, and APC-Cy7 anti-Gr-1 (RB6-8C5) from Thermo Scientific.