Quantifying Infected Myeloid Cells in Lungs

Analysis of infected myeloid cells in the lungs was performed as previously described (10 (link), 29 (link)). In short, lung tissue was homogenized in DMEM containing FBS using C-tubes (Miltenyi Biotec). Collagenase type IV/DNase I (Sigma-Aldrich) was added, and tissues were dissociated for 10 s on a GentleMACS system (Miltenyi Biotec). Lung tissue was then oscillated for 30 min at 37C. Following incubation, tissue was further dissociated for 30 s on a GentleMACS. Single cell suspensions were isolated following passage through a 40-μm filter. Cell suspensions were then washed in DMEM and aliquoted into 96-well plates for flow cytometry staining. Non-specific antibody binding was first blocked using Fc-Block. Cells were then stained with anti-GR1 Pacific Blue, anti-CD11b PE, anti-CD11c APC, and anti-CD45.2 PercP Cy5.5 (BioLegend). Live cells were identified using Zombie Aqua (BioLegend). No antibodies were used in the FITC (fluorescein isothiocyanate) channel to allow quantification of YFP⁺ Mtb in the tissues. All experiments contained a non-fluorescent H37Rv infection control to identify infected cells. Cells were stained for 30 min at room temperature and fixed in 1% paraformaldehyde for 60 min. All flow cytometry was run on a MACSQuant Analyzer 10 (Miltenyi Biotec) or an Attune Cytpix and was analyzed using FlowJo version 10 (Tree Star).