G418 sulphate

All experiments were performed with rat basophilic leukaemia cells (RBL-703/21-derived NFATp-DsRed-Express2) generated at The University of Nottingham^{16 (link)}. These are available from the authors for non-commercial purposes on the basis of a material transfer agreement (MTA). Cells were grown in Minimum Essential Medium Eagle’s (EMEM) cell culture medium supplemented with 10% v/v heat-inactivated foetal bovine serum (Gibco, UK), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (Merck, UK) in a 37 °C/5% CO₂ humidified cell incubator. To maintain stable expression of FcεRIα_H, cells were cultured with 1 mg/mL G418 sulphate (Thermo Fisher Scientific, UK) and 20 μg/mL Blasticidin S HCL (InvivoGen, USA) for selection of NFATp-DsRed-Express2 reporter gene expression.

AX4 cells and (Madin-Darby canine kidney (MDCK)-β-galactoside α2,6-sialyltransferase I (SIAT1) cells, which express higher amounts of six-linked sialic acids on their cell surface via exogenous expression of human SIAT1 (or ST6Gal I) [15 (link),16 (link)] were maintained in Eagle’s minimal essential medium (MEM) containing 10% fetal calf serum (FCS) and Dulbecco’s modified eagle medium (DMEM) containing 5% fetal calf serum and 1 mg/mL G418 sulphate (ThermoFisher Scientific, Tokyo, Japan), respectively. Both cell lines were incubated at 37 °C under 5% CO₂ and were passaged by the standard procedure.

B16-F10 was originally purchased from ATCC (CRL-6475) and genetically modified to express ovalbumin, eGFP, and neomycin phosphotransferase (Veliça et al., 2021 (link)). The resulting ovalbumin-expressing B16F10 cells were cultured in DMEM high glucose with pyruvate (11995065 Thermo Fisher) containing 0.75 mg/mL G418 sulphate (10131027, Thermo Fisher). HEK293 was a gift from Prof. Dantuma (Karolinska Institute, Stockholm) and cultured in DMEM high glucose with pyruvate. SKOV3 was purchased from ATCC (HTB-77) and cultured in McCoy’s 5A Medium (16600082, Thermo Fisher). Raji-GFP-Luc were purchased from Biocytogen (B-HCL-010) and cultured in complete RPMI (21875, Thermo Fisher). All media was supplemented with 1% penicillin streptomycin (15140122 Thermo Fisher) and 10% fetal bovine serum (FBS, A3160802 Thermo Fisher). Cell lines were frozen at low passage number (<5) in DMEM containing 10% DMSO and were typically passaged 3–4 times between thawing and experimental use. Cell line identities were confirmed by species-specific PCR analyses where relevant in non-human lines, as well as by functional properties. Otherwise cell lines were, as described above, obtained as authenticated lines from ATCC and were tested mycoplasma negative.