Rpmi 1640 glutamax 1 medium

Cell culture products (Hank’s balanced salt solution (HBSS), RPMI 1640 Glutamax I medium, Penicillin, Streptomycin, Trypsin) were obtained from Invitrogen (Cergy-Pontoise, France) while sera were obtained from Eurobio (Les Ulis, France) and Ultroser-G from PALL-Biosepra (Cergy-Pontoise, France).
Primary TEC cultures were obtained from mechanically minced fresh human thymus tissue and seeded onto cell culture flasks, as previously detailed (27 (link)). These culture conditions result mainly in medullary TECs (mTECS) that remain functional (27 (link)). Indeed, mTECs in primo-cultures keep their ability to express key molecules involved in immune tolerance processes such as autoimmune regulator, tissue-specific antigens, chemokines, and cytokines (27 (link)). After 8 to 12 days of primary culture, the confluent monolayers were Trypsinized and used in co-culture experiments, or frozen for further use.

Human peripheral blood mononuclear cells were isolated from buffy coats (obtained from Skåne University Hospital, Lund, Sweden) by Lymphoprep (ρ = 1.077 g/ml; Axis-Shield, Norway) density centrifugation at 700 × g for 20 min. Cells from the interphase were washed three times with phosphate-buffered saline (PBS) and monocytes were purified using anti-CD14-coated microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s instruction (monocyte purity was >96%). To obtain M1 or M2 macrophages, cells were cultured for 6 days in RPMI-1640-GlutaMAX-I medium (Life Technologies, UK), 10% (v/v) FBSi, 1% (v/v) AA, and 5 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, UK) or 12.5 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems) as described previously (18 (link)).

RPMI 1640 GlutaMAX™-I medium, fetal bovine serum (FBS), Alamar Blue cell viability reagent, and MicroBCA protein assay kit were obtained from Life Technologies (Carlsbad, CA, USA). Lumi-LightPLUS Western blotting kit (including anti-mouse IgG-HRP secondary antibody), trypsin–EDTA solution, and penicillin–streptomycin solution were purchased from Merck Group (Darmstadt, Germany). The 10% TGX SDS–PAGE stain-free precast gels and 2× concentrated Laemmli buffer were the products of Bio-Rad Laboratories (Hercules, CA, USA). LC–MS grade solvents were supplied by Supelco (Bellefonte, PA, USA). Other chemicals were of analytical grade and commercially available.

SEM (55 (link)), Jurkat, CEM, MOLT-16 and Nalm-6 cells (DSMZ) were cultured in RPMI 1640 GlutaMax-I medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific) (R10+). BHK-CD16a (FcγRIIIa V158) cells were cultured in R10+ medium supplemented with 10 μmol/l methotrexate (Sigma-Aldrich) and 500 μg/ml geneticin (Thermo Fisher Scientific) as described before (53 (link), 54 (link), 56 (link)). CHO-CD32a (FcγRIIa H131) cells were cultivated in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (57 (link)). Medium was supplemented with 1% NEAA (Thermo Fisher Scientific), 1% sodiumpyruvat (Thermo Fisher Scientific) and 500 µg/ml geneticin. Chinese hamster ovary cells (CHO-S, Thermo Fisher Scientific) were cultured in serum-free CD-CHO medium (Thermo Fisher Scientific) containing 1% HT supplement (Thermo Fisher Scientific) and 2 mM GlutaMax (Thermo Fisher Scientific). After transfection CHO-S cells were cultured in CD OptiCHO medium (Thermo Fisher Scientific) containing 1% HT supplement, 2 mM GlutaMax and 0.1% Pluronic F-68 (Thermo Fisher Scientific).

Endothelial Cell Growth Medium (cat. C-22010), penicillin/streptomycin solution, trypsin-EDTA solution, Lumi-Light PLUS Western Blotting Kit (including anti-mouse and anti-rabbit IgG-HRP secondary antibodies), protease and phosphatase inhibitor cocktails, extravidin-alkaline phosphatase (AP) conjugate, and 1-deoxymannojirimycin (DMJ) were purchased from Merck Group (Darmstadt, Germany). RPMI 1640 GlutaMAX™-I medium, DMEM medium, foetal bovine serum (FBS), Alamar Blue cell viability reagent, and MicroBCA Protein Assay kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The 2x concentrated Laemmli Buffer was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Biotinylated lectins: Phaseolus vulgaris leukoagglutinin (PHA-L), Phaseolus vulgaris erythroagglutinin (PHA-E), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Galanthus nivalis agglutinin (GNA), and Aleuria aurantia agglutinin (AAA) were purchased from Vector Laboratories Inc. (Burlingame, California, United States). ApogeeMix reference beads were purchased from Apogee Flow Systems (Northwood, UK). Polyvinyidene fluoride membranes (PVDF) were from Millipore (Burlington, MA, USA). All remaining chemicals were of analytical grade, commercially available.

Raji, Ramos, SK-BR-3 (DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and baby hamster kidney (BHK)-21 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were kept in RPMI 1640 Glutamax-I medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS; Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific; R10+ medium). BHK-21 cells that were co-transfected with plasmids encoding the FcεRI γ chain and either human FcγRIIIA 158F (BHK-CD16-158F) or FcγRIIIA 158V (BHK-CD16-158V) were cultured as described [38 (link)]. CHO glycosylation mutant Lec13 cells [39 (link),40 (link)] were maintained in MEM alpha medium with nucleosides (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% dialyzed FCS (Thermo Fisher Scientific, Waltham, MA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL). For culturing CHO-K1 and Lec13 cells transfected with antibody expression vectors, hygromycin B (Thermo Fisher Scientific, Waltham, MA, USA) was added to a concentration of 500 μg/mL. CHO cells stably transfected with a plasmid coding for the cDNA of human CD19 (Origene Technologies Inc., Rockville, MD, USA) were generated using standard procedures (Peipp, unpublished).

RPMI 1640 GlutaMAX™-I medium, fetal bovine serum (FBS), MicroBCA Protein Assay kit, Alamar Blue cell viability reagent, Acclaim PepMap trap (100 C18, 75 μm × 20 mm, 3 μm particle, 100 Å pore size) and analytical (Acclaim PepMap RSLC C18, 75 µm × 500 mm, 2 µm particle, 100 Å pore size) columns were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD63 mouse monoclonal primary antibody (clone RFAC4, cat. CBL553), Lumi-LightPLUS Western Blotting Kit (including anti-mouse IgG-HRP secondary antibody), Trypsin-EDTA solution, penicillin/streptomycin solution, SpeedBeads™ GE45152105050250 and GE65152105050250, and HEPES buffer were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal primary antibodies for Arf6 (clone 3A-1, cat. sc-7971) and HSP70 (clone C92F3A-5, cat. sc-66048) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Trypsin/Lys-C Mix was the product of Promega (Madison, WI, USA). All remaining chemicals were of analytical grade, commercially available.