Single cell suspensions of leukocytes were prepared from whole lungs by collagenase digestion for flow cytometry as described previously
13 (link). Briefly, each minced lung was incubated in 15 ml of complete media with 1 mg/ml collagenase (Boehringer Mannheim Biochemical, Chicago, IL), and 17 U/ml
DNase I (Sigma-Aldrich, St. Louis, MO) for 30 minutes at 37°C. The digested tissue was drawn through the bore of a 10-ml syringe repeatedly, and filtered through 100 µm mesh. Cells were stimulated with
PMA (0.05 mg/ml; Sigma-Aldrich, St. Louis, MO) and
ionomycin (0.75 mg/ml; Sigma-Aldrich, St. Louis, MO) for 4 h in the presence of
GolgiStop protein transport inhibitor (BD Pharmingen, San Jose, CA) before intracellular cytokine staining. An aliquot of 1 × 10
6 cells was first blocked by
anti-CD16/CD32 (Fc block; BD Pharmingen, San Jose, CA) antibodies and then stained using fluorochrome-conjugated antibodies against CD45, CD4,
IL-17A, IL-4 or IFN-γ (BD Pharmingen, San Jose, CA). For characterization of lung APCs, flow cytometry was performed using antibodies specific for CD11c (N418), CD103 (2E7), I-A
b (AF6-120.1), and
CD68 (FA-11), all from BioLegend (San Diego, CA);
CD11b (M1/70, BD Pharmingen); and
Ly6C (HK1.4) and F4/80 (BM8) from eBioscience (San Diego, CA). The FITC channel was used to detect autofluorescence of alveolar macrophages.
Zhou X., Loomis-King H., Gurczynski S.J., Wilke C.A., Konopka K.E., Ptaschinski C., Coomes S.M., Iwakura Y., van Dyk L.F., Lukacs N.W, & Moore B.B. (2015). Bone marrow transplantation alters lung antigen presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection. Mucosal immunology, 9(3), 610-620.
