Recombinant human stem cell factor

LCLs were cultured in upright non-adherent T25 tissue culture flasks (Nunc) at 37 °C and 5% CO₂. LCL media consisted of RPMI 1640 (Thermo Fisher, Waltham, MA, USA), supplemented with 1× GlutaMAX™ (Thermo Fisher), and 20% fetal bovine serum (Thermo Fisher). Erythroblasts were enriched in mixed PBL cultures following a previously published protocol [33 (link)]. Briefly, PBLs were cultured in erythroblast media containing StemSpan™ SFEM (Stem Cell Technologies, Vancouver, BC, Canada), 50 ng/mL recombinant human stem cell factor (R&D Systems, Minneapolis, MN, USA), 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL IGF1 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 ng/mL Interleukin 3 (Miltenyi Biotech), 2 U/mL human erythropoietin (R&D Systems), and 10 μg/mL Gentamicin (Thermo Fisher). Magnetic beads labelled with a CD71 antibody were then used to enrich for erythroblasts, following the manufacturer’s instructions (Miltenyi Biotec).

Laboratory of Allergic Disease 2 (LAD-2) mast cells were kindly provided by Dr. Arnold Kirshenbaum (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA). Cells were maintained in StemPro-34 medium (Life Technologies, Grand Island, NY, USA) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin, and 100 ng/mL recombinant human stem cell factor (R&D Systems, Minneapolis, MN, USA) as described previously^{43 (link)}.
LAD-2 cell degranulation was evaluated by the β-hexosaminidase release test⁴⁴ . Cells were treated for 16 h with cis- or trans-UCA with or without simultaneous sensitisation with biotinylated-IgE (100 ng/mL, BioPorto Diagnostics, Hellerup, Denmark) (IgE-mediated and non-IgE mediated degranulation), followed by 30 min of stimulation with streptavidin peroxidase (100 ng/mL) or calcium ionophore A23187 (1 µM, Sigma) in Tyrode’s buffer containing 0.1% BSA. Total β-hexosaminidase was obtained by lysing LAD-2 cells in 0.1% Triton X-100 in PBS. The supernatants were collected and incubated with an equal volume of p-nitrophenyl N-acetyl-β-D-glucosamide (Sigma, 4 mM in citrate buffer) for 1 h. The reactions were stopped by adding 0.4 M glycine buffer, and the signals were read at 405 nM. The percentage of degranulation was calculated as 100 × (OD stimulated − OD unstimulated)/(OD total lysate − OD unstimulated).

CECs were differentiated from human PBMCs according to the protocol by Heshusius et al.^{71 (link)} with modifications. Human PBMC were purified from buffy coats from healthy donors by density separation using Lymphoprep (STEMCELL Technologies). PBMC were seeded at 1 × 10⁶ cells/ml in erythroid differentiation-promoting medium based on StemSpan™ Serum-Free Expansion Medium (SFEM) supplemented with human recombinant EPO (2 U/ml, Roche), human recombinant stem cell factor (25 ng/ml, R&D Systems), dexamethasone (1 µM, Sigma-Aldrich), human recombinant insulin (10 ng/ml, Sigma-Aldrich), l-glutamine (2 mM, Sigma-Aldrich), iron-saturated holo-transferrin (20 μg/ml, Sigma-Aldrich), sodium pyruvate (1 mM, Gibco), MEM non-essential amino acids (1% v/v, Gibco), bovine serum albumin (0.1% m/v, Sigma-Aldrich), EmbryoMax Nucleosides (1% v/v, Merck), and 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). The expansion and differentiation of CECs were assessed by flow cytometry.