Flow cytometry
Bronchoalveolar lavage fluid (BALF) was performed via delivery of 0.8 mL of PBS intratracheally twice through a canula and centrifuged. Cells were immediately stained by antibodies. To obtain single-cell suspensions from lung tissues, lungs were intubated and digested for 1 hour at 37℃ with intermittent pipetting every 15 min in Hank’s Balanced Salt Solution(HBSS, Invitrogen) media containing 1mg/mL type 1A collagenase (Sigma) and 30μg/mL DNase I (Sigma) . Homogenates were passed through a 70 mm filter, treated with RBC lysis buffer, washed, and resuspended in PBS with 1% FBS. For Th cell differentiation analysis, cells were restimulated with 1X cell stimulation cocktail (plus protein transport inhibitors) (eBioscience) for 5 hours at 37℃ before surface and intracellular staining with appropriate cytokine antibodies. For surface staining, cells were incubated with antibodies cocktails for 30 min at 4℃. For intracellular staining, cells were fixed and permeabilized using Fixation/Permeabilization Buffer Set (eBioscience) according to manufacturer’s protocol. Fluorochrome-conjugated anti-mouse mAbs used in these experiments included the following: Fixable Viability Dye eF780, CD45-BV510, Siglec-F-AF647, CD11c-FITC, CD11b-PECY7, CD4-PECY7, IL-4-BV421, IL-17A-AF647, IFN-γ-FITC, IL-9-PE, CD44-FITC (Miltenyi), CD62L-APC, CD3-PERCPCY5.5. All antibodies were purchased from BD Biosciences unless otherwise specified. Cells were analyzed using Fortessa (BD) and FlowJo X software.