Cytofix cytoperm staining buffer kit

In vivo labelling was performed as described previously. Mice were intravenously injected with 2.5μg PE/Pacific Blue-conjugated anti-CD45 or anti-CD19 in PBS. After 4min mice were euthanized and lungs perfused with 10ml cold PBS through the right ventricle. Lungs were removed and roughly dissected with scissors before digestion in 1mg/ml collagenase D (Roche) and 10μg/ml DNAseI in RPMI for 45min at 37°C. Tissue was homogenised through a 70μm mesh and for experiments assessing BRM, lymphocytes were enriched by Ficoll-Paque density centrifugation (GE Healthcare). Cell suspensions were incubated with FC block in FACS buffer (2% FBS, 0.1% Sodium Azide, 1mM EDTA in PBS) for 15min and then stained in FACS buffer using predetermined optimal antibody concentrations for 30min. Cells were then washed and labelled with secondary labelling agents for 20min. For intracellular staining of antibody isotypes, Cytofix/Cytoperm Staining Buffer Kit (BD Biosciences) was used as per manufacturer’s instructions. Data acquisition was performed using a BD Fortessa X20 (BD Biosciences) and analysed using FlowJo v10.8 (Tree Star Inc.). For BRM cell phenotypic characterisation (Figures 1B and 1C), data acquisition was performed on a Cytek Aurora (Cytek Bioscienes). For cell sorting, samples were prepared as above and sorted using a FACSAria III (BD Biosciences).

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.