Cho s cells

Mouse neuroblastoma Neuro 2a cells and mouse lymphoma YAC-1 cells were obtained from the American Tissue Culture Collection (ATCC). Mouse neuroblastoma NXS2 cell line was given to us by Dr. H. N. Lode (Universitätsklinikum Greifswald, Greifswald, Germany). Human NK-92 cells transfected with CD-16 (RFcγIII) were a gift from Dr. B. Clemenceau (INSERM U. 892, Nantes, France) [16] (link). CHO-S cells obtained from Life Technologies (Saint-Aubin, France). NXS2 cells were grown at 37°C in 5% CO₂ in DMEM with 10% heat-inactivated fetal calf serum, 2 mM L-Glutamin, 100 units/mL penicilline, and 100 µg/mL streptomycin. Neuro-2a and YAC-1 cells were grown at 37°C in 5% CO₂ in RPMI 1640 with 10% heat-inactivated fetal calf serum, 2 mM L-Glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Human NK-92 cells transfected with CD-16 were grown at 37°C in 5% CO₂ in RPMI 1640 with 10% heat-inactivated fetal calf serum, 2 mM L-Glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 100 U/ml human recombinant IL-2. CHO-S cells were grown in suspension at 37°C in PowerCHO 2 serum-free CD medium (Lonza, Basel, Switzerland) supplemented with 4 mM L-Glutamine and ProHT reagent (Lonza).

CHO-S cells (Life Technologies # A11557-01) were maintained in Dulbecco’s modified Eagle’s medium + F12 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, arlsbad, CA, USA), 1% penicillin and streptomycin (Beyotime, Shanghai, China). The cells were cultured in a humidified atmosphere at 37 °C and 5% CO₂. The cells were plated at a concentration of 2 × 10⁵ cells/well in 24-well plates and allowed to attach overnight. On the second day, after reaching 80% confluence, the cells in each well were transfected with the vectors containing CMV, CMV mutant, CAG enhancer, CAG, CHEF-1α, mouse CMV, HEF-1α, or PGK using 1 μL Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) per 1 μg vector according to the manufacturer’s instructions. At 48 h post-transfection, G418 (800 μg/mL) was used to kill the un-transfected cell lines. Meanwhile, cells transfected with the different vectors were digested and reseeded at a concentration of 5 × 10⁴ cells/well in 12-well plates. Each cell type was plated into three wells.

HEK293T cells (Cat. No. CRL-11268), A-431 (Cat. No. CRL-1555) and BS-C-1 cells (Cat. No. CCL-26) were from ATCC and were cultivated in Dulbecco’s modified medium supplemented with 10% (v/v) fetal calf serum. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂, 95% air. Transient transfection of HEK293T cells was performed with TransIT-293 (Mirus) reagent according to the manufacturer’s protocol. CHO-S cells (Life Technologies, Cat. No. R80007) were maintained as shaking suspension culture using Power CHO 2CD medium (Lonza) supplemented with 8 mM L-glutamine, 0.1 mM hypoxanthine and 0.1 mM thymidine. Cells were seeded in DMEM with 10% fetal calf serum overnight to facilitate adhesion before transfection. Transient transfection was performed with Lipofectamine reagent (Invitrogen) according to the manufacturer’s protocol.

CHO‐S cells (#A11557‐01; Life Technologies, Carlsbad, CA, USA) were cultured in serum‐free medium in a humidified incubator for suspension culture at 37°C with 5% CO₂. The cells were plated at a density of 1.5 × 10⁵ cells/well in 24‐well plates. After about 24 hrs, the cells were transfected using Lipofectamine^® 3000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. About 48 hrs after transfection, CHO cells were collected and cultured in a culture medium supplemented with 800 μg/ml G418 (Invitrogen, Waltham, MA, USA) for 2 weeks. Subsequently, cell populations exhibiting stable transgene integration were cultured in CD CHO medium (#10743‐029; Life Technologies) supplemented with 8 mM l‐glutamine (#25030‐024; Life Technologies) in 125‐ml Corning shake flasks (#431255; Sigma‐Aldrich, St. Louis, MO, USA) with 30 ml of medium in the presence of 500 μg/ml G418 for 10–15 days; at 70–80% density, cells were collected for analysis.

Mouse IgG2a mAb 14G2a (γ2a, kappa) specific for GD2 was purchased from BD Biosciences (Franklin Lakes, NJ). Mouse IgG2a mAb 7H2, a gift from Dr. J. Portoukalian (Department of Dermatology, Edouard Herriot Hospital, University of Lyon, France) specific for O-acetyl-GD3, was used as a negative control. Mouse IgG2a anti-OAcGD2 mAb 8B6 was constructed by joining the complementary deoxyribonucleic acid for the variable region of the parental murine IgG3 antibody 8B6 [9 (link)] with the mouse constant regions of the γ2a heavy chain and the kappa light chain. Appropriate light and heavy expression vectors were co-transfected into chinese hamster ovary (CHO-S) cells (Life Technologies). The resulting antibody was affinity-purified from culture supernatant using the Hitrap rProtein A FF column (GE Healthcare Bios-Sciences, Uppsala, Sweden). The protein A affinity chromatography step was followed by anion-exchange chromatography on Sepharose Q for endotoxin removal. The purity of mAb preparations was verified by SDS-PAGE and size exclusion HPLC analyses. Endotoxin quantitation was evaluated using the LAL kinetic chromogenic assay (Lonza, Basel, Switzerland).