2 mercaptoethanol

Vero-hSLAM cells (74 (link)) (Vero cells [ATCC cat# CCL-81] with stable expression of human SLAMF1 receptor) were maintained in Dulbecco’s high-glucose modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), amphotericin-B (working concentration of 0.25 µg/mL, Thermo Fisher Scientific), and G418-geneticin (working concentration of 500 µg/mL). H358 cells (bronchioalveolar carcinoma cell line, ATCC cat# CRL-5807) were maintained in Roswell Park MEMorial Institute 1640 medium (RPMI 1640; Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum. HEp2 cells (epithelia carcinoma cell line, ATCC cat# CCL-23) were grown and maintained in minimum essential medium (MEM, Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum and 1% penicillin-streptomycin (working concentration of 50 µg/mL–50 U/mL, respectively). THP1 cells (acute monocytic leukemia cell line, ATCC cat# TIB-202) were grown and maintained in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum and 50 µM 2-mercaptoethanol (VWR Life Science, cat# M131).

Vero African green monkey kidney cells (ATCC, CCL-81, Manassas, VA, USA) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Quality Biological, 112-013,101CS, Gaithersburg, MD, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin and streptomycin antibiotics (Corning 30-003-CI, Corning, NY, USA), and 1% L-glutamine (Corning, 25-005-CI, Corning, NY, USA). Human small airway epithelial cells (HSAECs) were cultured with Ham’s F-12 GlutaMAX Supplement (ThermoFisher Scientific, 31765-035, Burlington, ON, Canada), 5% FBS, 1% L-glutamine, 1% penicillin and streptomycin, 1% non-essential amino acid solution (ThermoFisher Scientific, 11140-050, Grand Island, NY, USA), 1% sodium pyruvate (VWR, 45000-710, VA, USA) 0.01% 2-Mercaptoethanol (VWR Life Science, 76177-742, Radnor, PA, USA). All cells were incubated at 37 °C with 5% CO₂ supplementation.

The reagents and media were acquired as follows: Roswell Park Memorial Institute-1640 (RPMI-1640) medium with Ultraglutamine from Lonza (Verviers, Belgium); fetal bovine serum (FBS) from GE Health Care Life Sciences (Logan, UT, USA); 2-mercaptoethanol from VWR International (Leuven, Belgium); Dulbecco’s modified Eagle medium/F-12 nutrient mixture (Ham) (DMEM/F-12; 1:1) from Gibco (Paisley, UK); phosphate-buffered saline (PBS) from Fisher Reagent (Geel, Belgium); dimethyl sulfoxide (DMSO) and phosphoric acid from Merk (Darmstadt, Germany); dimethylformamide (DMF) from Romil (Cambridge, UK); uncoated ELISA kits from Invitrogen by Thermo Fisher Scientific (Vienna, Austria); TripleXtractor and RNA Kit (Blood and Cultured Cells) from GRiSP (Porto, Portugal); recombinant human IL-4 from R&D Systems (Minneapolis, MN, USA); and High Capacity RNA-to-cDNA Kit and Master Mix form Applied Biosystems (Foster City, CA, USA). When not specified, the reagents were from Sigma-Aldrich (St. Louis, MO, USA).

HEp-2 cells, verified to be contaminated with HeLa cells, were obtained from ATCC and were certified mycoplasma-negative and used acutely. OP9-R7FS cells were kindly provided by Juan Carlos Zúñiga-Pflücker (University of Toronto, Sunnybrook Research Institute) and authenticated by mouse STR profiling by ATCC and mycoplasma tested via MycoAlert Mycoplasma Detection Kit (Lonza). Cells were maintained at 37°C with 5% CO₂ in DMEM containing 10% heat-inactivated fetal bovine serum (FBS, Wisent), 100 U/mL penicillin and streptomycin (Gibco), and 50 µM 2-mercaptoethanol (Amresco).

Primary mouse thymocytes were isolated and formed into a single-cell suspension using a 70 µm cell strainer. Cells were labeled with 10 µm of CFSE and washed as described above. Cells were then cultured for 2 days in RPMI1640 containing 100 U/mL penicillin and streptomycin (Gibco) and 50 µM 2-mercaptoethanol (Amresco) and 5 µg/mL puromycin (Thermo Fisher Scientific) to induce apoptosis. Cultures were collected and washed with PBS; intact cells were pelleted by gentle centrifugation at 300× g for 5 min. Cell supernatants containing ApoB were transferred into mice by intravenous injection.

Daudi B cells were maintained at 37°C with 5% CO₂ in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin (Gibco), and 50 μM 2-mercaptoethanol (Amresco). Parental Daudi B cells and CD22-KO Daudi B cells were kindly provided by Dr. Joan Wither (Krembil Research Institute, Toronto).