Extended Data Fig. 2: Gating strategies for identifying granulocyte and myeloid cells by flow cytometry as well as cytokine/chemokine production in lungs.

Single-cell suspensions were prepared from the lungs of naïve animals i.v. injected with labeled panCD45 antibody prior euthanasia to identify cells located within the lung vasculature. Gating strategy was used to identify myeloid populations in Mtb-infected lungs. *DUMP: TCRβ, TCRγδ, NK1.1, B220; AM, alveolar macrophages, IM, interstitial macrophages; Eos, eosinophils; Neuts, neutrophils; Neuts iv-, parenchymal neutrophils. Anti-Siglec-F antibody was added to the panel in the same channel as the DUMP makers and analyzed in combination with F4/80 to isolate AM and EOS out from DUMP+ cells as showed in the FACS plot. (B-D) Sample FACS plot of (B) parenchymal macrophages gated on Live/CD45⁺/DUMP⁻/Ly6G⁻/CD45iv^neg/F4/80⁺CD64⁺ events, (C) total neutrophils (gated on Live/CD45⁺/DUMP⁻/CD11b⁺Ly6G⁺) and (D) parenchymal neutrophils (gated on Live/CD45⁺/DUMP⁻/CD11b⁺Ly6G⁺/CD45iv^neg) shown in the Fig. 3b,c. (E) Summary data of cytokine/chemokine levels in lung homogenates from Mtb⁻infected Bach1^-/- (n = 23) and WT (n = 26) at 28 days p.i. (pooled from five independent experiments; two-tailed, Mann–Whitney U-test). (F) Sample FACS plot of CD45.1 (WT) vs CD45.2 (KO) within AM, Ly6C⁺ IM, total and parenchymal neutrophils shown in the Fig. 3k–m. (G) Sample gating strategy used for the in vitro experiments with BMDMs. FACS plots shown in A-D and F-G are representative of at least two independent experiments performed. Each symbol represents an individual animal within the group. Significant differences are indicated with asterisks (ns, not significant).

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