Draq7

Single‐cell RNA‐seq experiment was performed by experimental personnel in the laboratory of GENECHEM. The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first washed with phosphate‐buffered saline (PBS), minced into small pieces (approximately 1 mm³) on ice and enzymatically digested with 100 U/mL collagenase IV (Worthington) and 30 U/mL DNase I (Worthington) for 45 minutes at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer and centrifuged at 300 g for 5 minutes. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were re‐suspended in PBS containing 0.04% BSA and re‐filtered through a 40 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein‐AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single‐cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).

Tissue was surgically minced on a laboratory sterile table, and tissue fragments were preserved in MACS tissue storage until processing.
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm³) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).

Mixed populations of human lung cells (obtained after mechanical and enzymatic digestion of lung specimens) were stained with fluorochrome-conjugated monoclonal antibodies against the following surface markers: CD4 (RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD19 (HIB19), CD45 (HI30), c-kit (104D2), FcεRI (AER-37), and CD326 (EBA-1). The antibodies were from BD Biosciences (Franklin Lakes, NJ, USA) or BioLegend (San Diego, CA, USA). To evaluate apoptosis/necrosis, mixed populations of human lung cells, human lung c-kit⁺ cells or primary human lung smooth muscle cells were treated with PBS or mefloquine and then stained with Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and DRAQ7™ (Biostatus Ltd., Shepshed, UK). To determine the effect of NAC on cell death, extracted lung cells were pre-incubated with or without NAC (8 mmol/L) for 2 h and subsequently treated with mefloquine or PBS for 24 h. The cells were stained for mast cell surface markers and Annexin V/DRAQ7 and analyzed on a LSR II or LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using the FlowJo software (TreeStar Inc., Ashland, OR, USA). In all flow cytometry analyses, doublet cells were excluded.

The brain tumour tissues were resected and kept in MACS tissue storage solution (Miltenyi Biotec). Tissue samples were washed with PBS (phosphate‐buffered saline), cut into small pieces on ice and digested with tumour dissociation kit (Miltenyi Biotec). Then, samples were passed through a 70 μm cell strainer and centrifuged for 5 min at 300 g. Red blood cell lysis buffer (Miltenyi Biotec) was used to lyse red blood cells in the pelleted cells. Cell pellets were washed with and re‐suspended in 0.04% BSA in PBS and passed through a 35 μm cell strainer. Single cells were then stained with Calcein‐AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences) to evaluate cell viability.