Gr1 clone rb6 8c5

After mice were euthanized, BALF was collected and centrifuged, and neutrophils were counted using the Kimura stain.
Intravascular and interstitial neutrophils in the lungs were distinguished by a flow cytometry-based method as previously described [19 (link)]. Briefly, an Alexa 633-labeled GR-1 antibody (clone RB6-8C5, staining kit: Invitrogen Corp., Carlsbad, CA, USA) was injected i.v. 5 min before euthanasia, labeling only intravascular neutrophils. After performing BAL, the inferior vena cava was dissected and non-adherent neutrophils were removed from the pulmonary vasculature by flushing 10 ml of PBS at 25 ml H₂O through the spontaneously beating right ventricle. Lungs were removed, minced, and digested with enzyme cocktail at 37°C for 60 min. A cell suspension was prepared by passing the digested lungs through a 70 mm cell strainer (BD Falcon, Bedford, MA, USA) which lysed the erythrocytes, and the remaining leukocytes were counted. The fraction of neutrophils in the suspension was determined by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Neutrophils were identified by their typical expression of CD45 (clone 30-F11, BD Biosciences-Pharmingen, San Diego, CA, USA) and GR-1 (clone RB6-8C5). The i.v. injected labeled GR-1 Ab differentiated intravascular and interstitial neutrophils.

Cell suspensions were analyzed for GFP expression or stained with antibodies against SSEA1 (FAB2155A, R&D Systems), Mac-1 (clone M1/70, BD Biosciences), or Gr1 (clone RB6-8C5, BD Biosciences). Cells were analyzed with an LSR II FACS (BD Biosciences) using Diva version 6.1.2 (BD Biosciences) and FlowJo software version 10.0.6

Bone marrow lineage negative (Lin^–), Sca-1⁺, c-kit⁺ (LSK) hematopoietic stem/progenitor cells were obtained from Csf2ra^KO mice³⁰ (link) according to SOPs (Table S5). In brief, bone marrow cells were obtained from 7 to 10-week-old Csf2ra^KO donor mice (n = 24, 12 males/12 females, body weight = 17.6–27.5 g) by removing and crushing the pelvic iliac crests, tibias, and femurs in IMDM media (Corning) containing 2% heat-inactivated fetal bovine serum (FBS), and 1% penicillin/streptomycin solution (Gibco). Mononuclear cells were hematopoietic lineage depleted with biotin-conjugated mouse lineage antibodies: mouse monoclonal CD5 (clone 53-7.3; BD Pharmingen), CD8a (clone 53-6.7; BD Pharmingen), CD45R/B220 (clone RA3-6B2; BD Pharmingen), CD11b (clone M1/70; BD Pharmingen), Gr-1 (clone RB6-8C5; BD Pharmingen), and TER-119 (BD Pharmingen) followed by magnetic bead separation (Dynabeads Sheep anti-Rat IgG, Invitrogen). After removing lineage-positive cells, the remaining cells were stained with 7-aminoactinomycin D (7-AAD) (Invitrogen), Streptavidin-FITC (fluorescein isothiocyanate), Sca-1 (clone D7)-PE (R-Phycoerythrin), and CD117/c-kit (clone 2B8)-APC (allophycocyanin) antibodies (BD Biosciences) and Lin⁻ c-Kit⁺ Sca-1⁺ cells were sorted using a FACS Aria II (BD Biosciences) gated on live cells (7-AAD negative).