Fix lyse solution

Cells in BALF were stained with fixable viability dye and anti-murine CD45-PE Fluor61 (eBioscience, San Diego, CA), SiglecF-Alexa647, CD11b-PE Cy7 (BD Bioscience, Franklin Lakes, NJ), CD11c-PerCP Cy5.5, Ly-6C AlexaFluor700 (BD Pharmingen, San Jose, CA), and Ly-6G-FITC (Biolegend, San Diego, CA). Cells were fixed and red blood cells were removed using fix/lyse solution according to manufacturer’s instruction (eBioscience, San Diego, CA). Quantification of total cell content was performed by addition of precision count beads (Biolegend, San Diego, CA) before measurement. Expression levels of activation markers were quantified via the median fluorescent intensity (MFI). Data were collected on a Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ) and analyzed using FlowJo software v10.7.1 (Treestar, Palo Alto, CA, USA). The full gating strategy can be found in Fig. S1.

Female C57BL/6 and 129S2 mice were co-housed one week prior to experiments. Mice were infected with low dose Mycobacterium tuberculosis (strain H37Rv, 200 CFU), using the Glas-Col inhalation exposure system. 24 h after infection, the amount of inhaled bacteria was controlled in five mice by plating the whole lung and counting CFU after 21 days of incubation at 37 °C. 7, 14, and 21 days after infection, blood and bone marrow from mice were analyzed by flow cytometry for progenitor frequency. Briefly, mice were lethally anesthetized with Ketamin/Xylazin i.p. and blood was readily collected from the inferior vena cava in syringes containing anti-coagulant (EDTA). Bone marrow was collected by flushing femoral bones with RPMI1640 media. Cells were resuspended in FACS buffer (1% FBS, 2 mM EDTA), blocked for unspecific binding with rat-IgG and anti-FcR antibodies and stained in the dark for 30 min at 4 °C. Details on antibodies used are presented in the section: Surface and intracellular staining. Finally, samples were fixed in 4% PFA for 30 min at room temperature (RT). Staining of blood cells was performed in whole blood. Following blocking and antibody incubation, it was transferred into fix/lyse solution (eBioscience) for 30 min at RT. Cells were acquired on the CytoFLEX S (Beckman Coulter) flow cytometer and analyzed with the FlowJo software (Treestar).

To investigate the phenotype of neutrophils, freshly isolated NDGs and LDGs were incubated with the following antibodies for 30 minutes on ice: CD10-PerCP-Cy5.5 (clone HI10a, BD Pharmingen, 25μg/mL), CD15-PE-Vio770 (clone VIMC6), CD16-VioGreen (clone REA423), CD33-FITC (clone REA775), CD62L-VioBlue (clone 145/15), CD63-PE (clone REA1055) and CD66b-APC (clone REA306), all Miltenyi Biotec, Germany. Flow cytometry was performed using the MACSQuant® flow cytometer (Miltenyi Biotec, Germany). Data analysis was performed with FlowJo software (TreeStar, CA, version 10.6.1). To determine the absolute numbers of NDGs from peripheral blood, Truecount TM tubes (BD Biosciences) were used according to the manufacturer’s instructions after staining full blood with CD45-FITC (clone REA747), CD16-VioGreen (clone REA423) and CD19-APC (clone REA675, all Miltenyi Biotec) and lysis with immediate fixation using the Fix/Lyse Solution (Invitrogen). In contrast, absolute numbers of LDGs were determined by calculating the percentage of CD15⁺ LDGs contained in the PBMC-layer from the absolute number of CD45⁺ PBMCs as determined by Truecount assessment.