Live dead fix blue

lung tissue was enzymatically digested/dissociated into single-cell suspension using mouse “Lung Dissociation Kit” and “gentleMACS Octo Dissociator with Heaters” both from Miltenyi Biotech Inc. as per the manufacturer’s instructions. In brief—lungs were washed twice with cold PBS, cut into small pieces, transferred to MACS ‘C Tube’ containing the enzyme mix. The tube then loaded on the gentleMACS instrument and run the lung-specific program. The dissociated cell suspension was passed through a 70-µm cell strainer and washed three times with cold PBS containing 1% FBS and 0.2% BSA.
Cells were washed three timed with protein-free PBS and stained with viability dye (Live/DEAD fix blue, Thermo Fisher Scientific) in the dark, cells were washed again and incubated with FcBlock (anti-mouse CD16/CD32) in FACS buffer, followed by staining with fluorochrome-conjugated antibodies for Lung Panel (antibodies, clones, fluorochromes, and manufacturers were described in Supplementary Table 1). Fluorescence minus one controls were used to set the gates to select the positive population. Lung AΦs were identified as CD64⁺CD11c^highCD11b^lo (or by CD11c^highSiglec-F^high) and iΦs as CD64⁺CD11c^lowCD11b^hi (Supplementary Fig. 4)^{2 (link)}.

The reagents were purchased as follows: red blood cell lysis buffer from Sigma-Aldrich (St. Louis, MO, United States); Percoll, phosphate-buffered saline (PBS) and fetal bovine serum (FBS) from Fisher Scientific; fixation solution from Biolegend.
The following anti-mouse monoclonal antibodies were purchased from BioLegend (San Diego, CA, United States): CD3 APC-Cy7 (17A2), CD4 Brilliant Violet 605 (GK1.5), CD8a PE-Cy7 (53-6.7), CD19 FITC (6D5), TCRγδ PE (GL3), and F4/80 Brilliant Violet 510 (BM8). The following anti-mouse monoclonal antibodies were purchased from BD Biosciences (San Diego, CA, United States): CD11c APC-R700 (N418). The following anti-mouse monoclonal antibodies were purchased from Bio-Rad (Hercules, CA, United States): Ly6B.2 Alexa Fluor 647 (7/4). The following anti-mouse monoclonal antibodies were purchased from Thermo Fisher Scientific: Live/Dead Fix Blue (L23105).

Total lung cells isolated above were stained with Live/Dead Fix Blue (L34961, Thermofisher scientific, Waltham MA), F4/80, CD64, CD11b (123107, 139313, 101223, Biolegend, San Diego, CA), SigletF (740388, BD Biosciences, San Jose, CA), then permeabilized and fixed using Cytofix/Cytoperm Fixation/Permeablization Kit (554714, BD Biosciences, San Jose, CA), and stained for MMP12 (sc-390863 AF647, Santa Cruz, Dallas, TX) (S4 Fig).