Apc conjugated anti f4 80 clone bm8

Tumors were harvested from donor mice and dissociated using Tissue and Tumor Dissociation Reagent (661563, BD Bioscience, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. After passing through a 70 μm cell strainer, the single-cell suspensions were pretreated with Fc Block (anti-CD16/32 clone 2.4G2; BioXcell, Lebanon, NH, USA) and stained with FITC-conjugated anti-CD45 (clone 30-F11, BioLegend), PerCP/ Cy5.5-conjugated anti-CD11b (clone M1/70, BioLegend, San Diego, CA, USA), PE/Cy7-conjugated anti-Ly6C (clone HK1.4, BioLegend, San Diego, CA, USA), APC-conjugated anti-F4/80 (clone BM8, BioLegend, San Diego, CA, USA), Pacific Blue-conjugated anti-Gr1 (clone RB6-8C5, BioLegend, San Diego, CA, USA) antibodies, and propidium iodide (P4864, Sigma-Aldrich, St. Louis, Missouri, USA). Each target cell was sorted using SH800S (SONY, Tokyo, Japan). The data were analyzed using the FlowJo software V.10.6.2 (BD Biosciences, Franklin Lakes, NJ, USA).

After the mice were euthanized by ketamine-xylazine overdose, a plastic cannula was inserted into the trachea. Bronchoalveolar lavage samples were obtained by thrice washing each pair of lungs with 1.0-ml aliquots of 0.9% saline. Following centrifugation, the bronchoalveolar lavage cell pellets were washed and resuspended in phosphate buffered saline (PBS), and the total cell counts were determined using a portion of the suspension. The cytospin preparations were fixed and stained using Diff-Quick (Dade, Behring, Deerfield, IL, USA), and the differential cell counts were analyzed. In the remaining suspension, erythrocytes were lysed using red blood cell lysis buffer. The remaining cells were washed and incubated with anti-CD16/32 in order to block the Fc receptors (Tonbo Bioscience, San Diego, CA, USA). The following antibodies were used to stain the cell suspensions: FITC-conjugated anti-Ly-6G (clone 1A8; BD Biosciences, Franklin Lakes, NJ, USA), PE-conjugated anti-Siglec-F (clone E50-2440; BD Biosciences, Franklin Lakes, NJ, USA), and APC-conjugated anti-F4/80 (clone BM8; BioLegend, San Diego, CA, USA). BD FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the cell profiles of the BAL fluids.