Facs aria fusion cytometer

Flow cytometry and FACS were performed as described previously [27 (link),29 ]. Briefly, cells isolated from lung tissue as described above were pelleted and resuspended at 10⁷ cells/mL in PBS with 0.1% BSA. Non-specific antibody binding was blocked with anti-CD16/CD32 followed by a master mix antibody cocktail (50 μL per 10⁷ cells) that included 10 μL of each of the following antibodies: anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD11c-AF700 (clone N418; eBioscience), anti-Gr1 (Ly-6G and Ly-6C)-APC (clone RB6-8C5; BD Biosciences), anti-Siglec F-PE (clone E50-2440; BD Biosciences) and anti-MHCII (I-A/I-E)-PE-CY-7 (clone M5/114.15.2; eBioscience). Cells were incubated with antibodies at 4°C for 30 min while protected from light, then pelleted and resuspended in PBS with 0.5% BSA at 10⁸ cells/mL. Samples were evaluated and sorted using a FACSAria Fusion cytometer (BD Biosciences). Eosinophils were identified as CD45⁺/CD11c^-/GR1^lo/MHCII^-/Siglec F⁺ cells. Additional details are provided in S1 Appendix.

Lung ILCs were enriched from lung single-cell suspensions by using the EasySep™ Mouse ILC2 Enrichment Kit (StemCell). After enrichment, cells were stained with lineage mAb (CD3, CD4, CD8α, TCRαβ, TCRγδ, CD19, B220, CD11b, CD11c, F4/80, TER119, FcεRIa, CD49b, Ly6G) and ILC markers (CD90.2, CD45.2, NK1.1, ST2, IL-18Rα, CD49a). ILC2 were purified as Lin⁻CD45.2⁺CD90.2⁺NK1.1⁻ST2⁺IL-18Rα^+/- ILC1-like were purified as Lin⁻CD45.2⁺CD90.2⁺NK1.1⁻ST2⁻CD49a⁺IL-18Rα⁺. Cells were sorted using a FACSAria Fusion cytometer (BD, France).

LSK cells were purified as described previously (61 (link)). In brief, BM cells were isolated from femora and tibiae of mice in PBS containing 0.5% BSA and 2 mM EDTA buffer. Lineage negative cells (Lin-) were isolated using the Lineage Cell Depletion Kit (Miltenyi Biotec). Lin- cells were stained with APC-c-Kit and PE-Sca1 antibodies, and LSK cells were purified with a BD FACSAria Fusion cytometer and cultured in SFEM medium plus 10% FBS supplemented with 20 ng/mL Flt3L, 20 ng/mL IL-6, 100 ng/mL SCF, 20 ng/mL TPO, and 0.1 mM β-ME. After 48 hours of culture, LSK cells were spin-infected with retroviruses expressing WT or Q61R mutant NRAS preloaded on RetroNectin- coated plates, with 10 μg/ml polybrene (Sigma-Aldrich). Cells were either transplanted 24 hours after infection or sorted for GFP positivity 48 hours after infection for cell proliferation assay, colony assay, or biochemical experiments.

HH-12 chicken embryos were electroporated with shCONTROL or shADSL plasmids, and 48 hr post electroporation (hpe), a single-cell suspension was obtained by digestion for 10–15 min with Trypsin-EDTA (MilliporeSigma) and labeled with Hoechst and α-ELAVL3/4 antibody used with Alexa 647-conjugaded anti-mouse secondary antibody. Alexa 647, Hoechst, and GFP fluorescence were determined by FACSAria Fusion cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star) and Multicycle software (Phoenix Flow Systems; cell cycle profile analysis).

For flow cytometry, Meflin-CreER^T2; LSL-tdTomato mice were sacrificed after i.p. administration of 0.1 mg/g TAM performed every other day for a total of 10 times, and then their kidneys were harvested. The kidneys were minced and digested with a digestion buffer (1 mg/mL type 1 collagenase, 0.1 mg/mL DNAse in Hank’s balanced salt solution) to obtain single cell suspensions. Following treatment with a red blood cell (RBC) lysis buffer (BioLegend), the cell suspensions were plated onto 96-well plates and incubated with the following antibodies for 30 min: anti-CD45-Pacific Blue (cat. no. 103126, clone 30-F11; BioLegend), anti-CD31-PE/Cy7 (cat. no. 102418, clone 390; BioLegend), anti-CD146-FITC (cat. no. 134706, clone ME-9F1; BioLegend), anti-CD140a-Alexa647 (cat. no. 562777, clone APA5; BD Biosciences, Franklin Lakes, NJ), anti-CD140b-Biotin (cat. no. 136010, clone APB5; BioLegend), and anti-Sca1-Brilliant Violet 605 (cat. no. 108134, clone D7; BioLegend). Flow cytometry was performed using a FACSAria Fusion cytometer (BD Biosciences), followed by data analysis using FlowJo software (Tree Star Inc., Ashland, OR).