Mil 3

Bone marrow from the femurs and tibias of wild-type C57BL/6 mice or Siglec-8 transgenic mice (16 (link)) was cultured in complete medium (DMEM supplemented with 10% FBS (Cytiva), 2 mM L-glutamine (Gibco), 1% antibiotic-antimycotic solution (Gibco), 50 µM β-mercaptoethanol (Gibco) in the presence of 10ng/mL mIL-3, Stemcell Technologies). After 2 days of culture, cells in suspension were transferred to a new flask and fresh culture medium was added two times per week with the cell count maintained between 0.5-1x10⁶/mL for 6-8 weeks prior to use. MC maturity was monitored using flow cytometry staining with antibodies against CD45 (clone 30-F11, Biolegend), and MC markers CD117 (clone 2B8, eBiosciences) and FcεRIα (clone MAR-1, Biolegend). Siglec-8 expression was monitored using PE conjugated antibody from R&D Systems (clone 837535). Bone marrow mast cells are referred to as BMMC (from C57BL/6 wild-type mice) and S8-BMMC (from Siglec-8 transgenic mice).
Methods for human MC generation and activation can be found in the Supplementary Material.

At day 2 post-infection, Sca1⁺/lin^- cells were plated at 5 x 10³ cells/mL in methyl-cellulose supplemented with mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL)(Stem-Cell Technologies, Vancouver, Canada). On day 10 after plating, the number of colony forming units (CFUs) was determined. After washing out from the methyl-cellulose, the cells were stained with specific antibodies for the detection of surface marker expression by FACS. 5 x 10³ cells/ml were replated in methyl-cellulose, thus permitting determination of the serial replating potential.

To assess clonogenic progenitor frequencies, 3 × 10⁴ whole marrow cells, 5 × 10² 5-fluorouracil-enriched marrow cells or 3 × 10² sorted Lin⁻c-Kit⁺ cells were plated in methylcellulose containing m-IL-3, h-IL-6, h-erythropoietin, and m-SCF (M3434; STEMCELL Technologies). Where indicated, cells were treated with 10 µM SMAD3 inhibitor SIS3 (Millipore) or dimethyl sulfoxide vehicle. Colonies were scored 8–10 days later. For replating assays, marrow cells were scraped off using a cell scraper and collected in a conical tube containing PBS. Cells were washed and counted before replating onto fresh methylcellulose in equal proportions per condition, and normalized to the number of input cells.

CFU-GEMM, CFU-GM and BFU-E colony formation was assessed in vitro using washed suspensions of 5 X10⁴ marrow cells or 3 X 10⁵ spleen cells suspended in IMDM and plated in 1% methylcellulose with 30% FBS (Methocult medium, catalogue #3534, STEMCELL Technologies, Vancouver, BC Canada) containing 10 ng/μL of mIL3 (catalogue #02733, STEMCELL), 10 ng/μL of rhIL6 (#206-IL-010, R&D SYSTEMS, Minneapolis, MNUSA), 50ng/μL of THPO (#288-TP-005, R&D) and 3 U/μL of rhEPO (Johnson and Johnson, New Brunswick, NJ USA). All colony-forming assays were performed between weeks 15–18. Colony-forming assays were performed in triplicate with at least 3 replicate experiments and colony formation was assessed at 7 days. CFU-Mk colony formation was assessed using 5 X10⁴ washed marrow cells or 3 X 10⁵ spleen cells suspended in IMDM and plated in methylcellulose (Megacult-C medium, # 04850, STEMCELL) containing 10 ng/μL of mIL3, 10 ng/μL of rhIL6 and 50ng/μL of THPO, R&D). Colony number was assessed at 7 days after staining with acetylcholinesterase activity as described in the Megacult-C protocol for murine cells.

32Dcl3 (here after named as 32D) and BA/F3 cells (ACC-411, ACC-300, DSMZ, 2018–01) were cultured as described previously [25 (link), 26 (link)]. TF-1 cells (ACC-334, DSMZ, 2018–01) were cultured using RPMI 1640/10%FCS/GM-CSF (5 ng/ml). All cell lines were routinely tested for mycoplasma using PCR. Authentication of cell lines was performed using qRT-PCR for murine or human housekeeping gene as well as cell surface expression of characteristic receptor expression pattern (CD34, CD11b, Gr-1) using FACS analysis. Primary murine cells were cultured in serum-free BIT9500 cell culture medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with mIL-3 (10 ng/ml), mIL-6 (5 ng/ml) and mSCF (50 ng/ml). All cytokines were purchased form ImmunoTools, (Friesoythe, Germany). Further, lineage negative transgenic SCLtTA/Bcr-Abl BM cells were retrovirally infected using MSCV-ER-Hoxb8-Neo plasmid as described previously [27 (link)]. ER-HoxB8 derived immortalized progenitor cells were cultured in IMDM containing 10% FBS, 5% SCF-supernatant and 1% Pen-Strep and selected with G418 (1 mg/ml) for 1 week. FACS analysis for Gr-1, CD11b, B220, CD3 and Ter119 (BioLegend, Fell, Germany) were performed demonstrated the absence of mature cell surface markers.