Lung digestion was performed as previously described (MacLean et al., 2022 (link)). Briefly, mice were in vivo labeled with i.v. anti-CD45 antibodies 4 min before euthanization. Lungs were perfused with PBS and roughly dissected before incubation in collagenase D (1 mg/ml, Cat# 11088858001; Roche) and DNaseI (10 μg/ml, Cat# DN25; Merck) at 37°C for 45 min. Tissue was homogenized through a 70-μm filter and stained in FACS buffer (2% FBS, 0.1% sodium azide, 1 mM EDTA in PBS). Fragment crystallizable regions of an antibody block was performed for 15 min and staining for 30 min at 4°C. Cytofix/Cytoperm Staining Buffer Kit (BD Biosciences) was used for intracellular staining of antibody isotypes. Data acquisition was performed using a BD Fortessa X20 (BD Biosciences) and analyzed using FlowJo v10.8 (Tree Star, Inc.).
For intracellular antibody staining of IFNγ, cells were digested as above, resuspended in RPMI with 10 μg/ml brefeldin A, and incubated at 37°C for 3 h. Cells were subsequently processed as detailed above for antibody isotype staining.
For intracellular antibody staining of IFNγ, cells were digested as above, resuspended in RPMI with 10 μg/ml brefeldin A, and incubated at 37°C for 3 h. Cells were subsequently processed as detailed above for antibody isotype staining.
Free full text: Click here
