Anti cd45 2 percp cy5

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).