Igg2aκ apc

Cells were blocked with normal sheep serum and stained with monoclonal anti-mouse CD4 antibodies (RM4-5 clone; eBiosciences; San Diego, CA, USA). Intracellular staining of CD4 T cells was performed using anti-mouse IL-10-PE antibody (JES5-16E3 clone), anti-IL-4 PECy7 (11B11 clone), and anti-IL-17 eFluor 660 (eBio17B7 clone) or the respective controls IgG2bκ PE, IgG1κ PECy7, and IgG2aκ eFluor 660 (all against mouse proteins, from eBioscience). Treg cells were characterized by staining for CD4, CD25, and Foxp3, according to the manufacturer's instructions (Mouse Treg Staining Kit, eBiosciences).
Pulmonary myeloid dendritic cells (CD11c⁺CD11b⁺Gr1⁻B220⁻) were characterized as previously [26 (link)], using anti-Gr1 FITC (RB6-8C5 clone) anti-CD11c PE (N418 clone), anti-CD11b PEcy7 (M1/70 clone), and anti-B220 APC (RA3-6B2 clone) or the respective controls IgG2bκ FITC, IgG armenian hamster PE, IgG2bκ PECy7, and IgG2aκ APC (all against mouse proteins, from eBiosciences).
All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using the FlowJo X 10 and 7.6.5 software (Tree Star Inc., Ashland, OR, USA). Percentages of total cells were presented in bar graphs.

Eight-week-old mice were injected with saline or 4 mg/kg BLM, intratracheally. Mice were sacrificed 7 days after BLM injection, and saline perfused lungs were treated using a mouse lung dissociation kit (Miltenyi Biotec, Auburn, CA, USA), in the gentleMACS^TM Dissociator (Miltenyi Biotec). Red blood cells (RBCs) were lysed with RBC lysis solution (Miltenyi Biotec), and isolated cells from the mouse lung were double stained with anti-CCSP/anti-EpCAM for club cells or anti-CD74/anti-EpCAM for AT2 cells. Isotype control antibodies (IgG2a κ-FITC and IgG2a κ-APC; eBioscience, San Diego, CA) were used to establish gating parameters for positively stained cells. Double-positive populations were sorted using a BD FACSAria^TM III cell sorter (BD Biosciences, San Jose, CA, USA). Sorted double-positive cells were subjected to an RNeasy Micro Kit (Qiagen, Germany) to obtain mRNA for qRT-PCR assays or plated on chamber slides (SPL Life Sciences, Korea) for in situ proximity ligation assay (PLA) analysis.