Cd45 2 percp cy5

Lung cells were obtained from digesting masticated lungs in a solution of 5% fetal bovine serum, 1 mg/mL collagenase type IV, and 0.02 mg/mL DNase I (Sigma-Aldrich) in RPMI 1640 medium for 45 minutes at 37^oC. Peripheral blood mononucleocytes (PBMCs) were isolated from blood drawn from the right jugular. Red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend). For quantifying the number of PACs in the lungs and peripheral blood, both PBMCs and lung cells were stained with anti- Ter119-Pacific Blue, CD11b-redFuor (Tonbo Biosciences, catalog #10141–234 and #80–0112), CD31-PECy7 (BioLegend, catalog #102524), and DAPI. To quantify engraftment, PBMCs were stained with anti-Ter119-Pacific Blue, CD45.1-PE (BD Biosciences, catalog #561872), CD45.2-PerCPCy5.5 (Tonbo Biosciences, catalog #65–0454), and DAPI. Flow cytometry was performed using a BD LSRFortessa and the data analyzed using FlowJo. Antibody-conjugated beads (Thermo Fisher) were used for single stain compensation controls. Unstained cells and fluorescence-minus-one controls were used for gating populations in all flow cytometry experiments.

Cells were resuspended at a concentration of 10⁶ cells/100 μl fluorescence-activated cell sorting (FACS) buffer (1X PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (BD Biosciences) for fifteen minutes, stained with antibody-fluorophores for one hour on ice, then washed with ten volumes of FACS buffer. The following antibodies were used: B220-PE-Cy7 (RA3–6B2, Tonbo), CD11b-APC-Cy7 (M1/70, Tonbo), CD45.1-FITC (A20, Tonbo), CD45.2-PerCP-Cy5.5 (104, Tonbo), CD90.2-APC-Cy7 (30-H12, BioLegend), F4/80-APC-Cy7 (BM8, BioLegend), CD138-BV711 (281–2, BD), CD21-APC (B-ly4, BD), CD43-PE (S7, BD), GL7-eFluor660 (GL-7, eBioscience), CD23-eFluor450 (B3B4, eBioscience), IgM-FITC (II/41, eBioscience), Annexin V-APC (kit number 88–8007-72, eBioscience), and Zombie Yellow Fixable Viability Dye (kit number 423104, BioLegend). An LSRII was used for analysis and an ARIAII was used for sorting (BD Biosciences). All flow cytometry data was analyzed with FlowJo version 9.9.5. Preceding all flow cytometry analyses presented is the following gating strategy: 1) lymphocytes (forward scatter [FSC]-area by side scatter [SSC]-area), 2) singlets (FSC-width by FSC-height), 3) singlets (SSC-width by SSC-height), 4) live cells (viability dye⁻), 5) exclusion of non-B cell lineage cells (Thy1.1⁻F4/80⁻CD11b⁻).