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Gibco freestyle 293 expression medium

Manufactured by Thermo Fisher Scientific
Sourced in United States

GIBCO™ FreeStyle™ 293 Expression Medium is a serum-free, chemically defined media designed for the transient transfection and expression of recombinant proteins in 293 cell lines.

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19 protocols using gibco freestyle 293 expression medium

1

Purification of Recombinant Human FcRn

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Recombinant human FcRn was cloned into a proprietary mammalian expression vector encoding both the human α (FCGRT) and β (β2 microglobulin)-chains. A second expression vector was made with the C terminus of the α-chain appended with a dual AviTag® and His6 tag for in vivo biotinylation and purification purposes, respectively. HEK 293 cells (proprietary strain) were transfected using 293fectin (Invitrogen 12347019) and standard protocols. Cells were fed with both Gibco® FreeStyleTM 293 expression medium (Invitrogen catalog no. 12338018) and a proprietary in-house feed. For in vivo biotinylation, huFcRn was co-transfected with BirA, and media were supplemented with 100 μm biotin, 10 μm ATP, and 100 μm Mg2+. Conditioned medium was harvested 10 days post-transfection and adjusted to pH 5.8 for purification over IgG-Sepharose 6 Fast Flow resin (GE Healthcare catalog no. 17-0969-01) as previously described (24 (link)). Purified huFcRn was dialyzed into PBS, pH 5.5, at 4 °C for 24 h and analyzed by SDS-PAGE and HPLC-SEC to confirm purity.
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2

Purification of Porcine CCL Proteins

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The established HEK 293 cells stably expressing porcine CCL proteins were harvested and resuspended in Gibco® FreeStyleTM 293 expression medium (Invitrogen), and incubated in the CELLSPIN system (INTERGRA bioscience, NH, USA) for three to four days. Supernatant was collected, centrifuged at 3000 rpm for 20 min, filtered through a 0.22 μm pore size membrane, and purified using HisPur cobalt resin (Invitrogen) with Econo-Column® chromatography (Bio-Rad laboratories, Hercules, CA, USA) following the manufacturer’s protocol. The eluted proteins were concentrated using Amicon® Ultra-15 (molecular weight cut-off 3 kDa, Millipore Corp., Bedford, MA, USA), mixed with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals, Laval, QC, Canada), and kept at −20 °C for further experiments.
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3

Characterization of Engineered Mouse Cell Lines

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MILE SVEN 1 (ATCC) mouse endothelial cell line engineered to express DsRed and firefly luciferase, denoted MS1, has been described previously [35 (link), 36 ]. MS1 was used for subsequent generation of the MS1-TEM1 cell line that expresses human TEM1 (hTEM1) and EmGFP, in addition to DsRed and fLuc [35 (link), 36 ]. 2H11 murine endothelial cell line was used as muTEM1-positive control (ATCC). TC1, a C57BL/6 mouse lung adenocarcinoma cell line transformed with HPV-16 E6 and E7, was a generous gift from Dr Yvonne Patterson (University of Pennsylvania). The TC1 cell line engineered to express firefly luciferase fLuc, denoted TC1, was used in this study. MS1, MS1-TEM1, 2H11 and TC1 cells were cultured in RPMI1640 medium (Corning Cellgro, USA) containing 10% fetal bovine serum (FBS), 100 I.U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5 % CO2. 293-F cells were cultured in Gibco® FreeStyle™ 293 Expression Medium (Invitrogen) in a shaker flask and incubated in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 125rpm.
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4

Efficient Production of Recombinant rIL4-10 Fusion Protein

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rIL4-10 FP was produced by transient transfection of HEK293F cells with a
pcDNA3.1-neo expression vector (Invitrogen, Carlsbad, CA, USA) containing a
dual CMV (cytomegalovirus) promoter. The vector contained 2 transgenes: cDNA
coding for rIL4-10 and cDNA coding β-galactosidealpha-2,3-sialyltransferase
to optimize glycan capping with sialic acid. To enable purification, a
hexa-histidine affinity tag was cloned on the N-terminus of rIL4-10 FP.
Cells were cultured in GIBCO FreeStyle 293 Expression Medium (Invitrogen).
The medium contained no serum or antibiotics. Cells were grown in flasks on
a shaker platform in humidified, 5% CO2 cell culture incubator at
37 °C. Cells were split 3 to 4 times prior to transfection and they were
transfected at the cell viability of 90% and 1 million cells/mL cell
density. The transfection reagent used was 293fectin (Invitrogen). Culture
supernatant was harvested 72 hours after transfection.
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5

Transient Transfection of Recombinant Proteins

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Soluble recombinant proteins were produced by transient transfection of HEK293-6E mammalian cells grown in Gibco FreeStyle 293 expression medium (catalog no. 12338026, Thermo Fisher Scientific) as previously described (41 (link),42 (link)). Prey proteins were produced by transfecting 0.5 μg of DNA per million cells, and the conditioned medium was harvested 5 days after transfection. Prey protein expression levels were normalized using their β-lactamase activity to levels required for the AVEXIS assay, essentially as described (41 (link)). For the expression of biotinylated bait proteins, the bait plasmid was cotransfected with a plasmid encoding a secreted version of the biotin-ligase (BirA) (43 (link)) and dialyzed overnight in phosphate-buffered saline (PBS) to remove the unconjugated d-biotin in the medium. Baits were normalized by a standard ELISA using a monoclonal antibody (OX68) recognizing the Cd4d3+4 tag (32 (link)).
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6

Cell Culture Protocols for Various Cell Lines

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Human osteosarcoma U2OS and embryonic kidney fibroblast 293T cells obtained from ATCC were cultured in modified McCoy’s 5A medium (Thermo Fisher) and high-glucose Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% GlutaMAX (Thermo Fisher), and 1% penicillin-streptomycin (Pen-Strep; Thermo Fisher), respectively. The grivet kidney Vero cells (from ATCC) were cultured in DMEM supplemented with 2% FBS, 1% GlutaMAX, and 1% Pen-Strep. Human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured in EGM medium supplemented with EGM-SingleQuots (Lonza). Huh-7.5.1 cells were cultured as described above for 293T cells, with the addition of 1% nonessential amino acids. All adherent cell lines were maintained in a humidified 37˚C, 5% CO2 incubator. Freestyle-293-F suspension-adapted HEK-293 cells (Thermo Fisher) were maintained in GIBCO FreeStyle 293 expression medium (Thermo Fisher) using shaker flasks at 115 rpm, 37˚C, and 8% CO2.
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7

Crystallization of Lipid-Binding Proteins

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pNPB was purchased from Sigma-Aldrich (St. Louis, MO) and a 5.6 M stock solution was prepared in chloroform. SYPRO Orange was purchased from ThermoFisher (Waltham, MA). Crystallization screens Classics Lite Suite and JCSG+ Suite were purchased from Qiagen (Venlo, Netherlands) and Index HT from Hampton Research (Aliso Viejo, CA). Gibco Freestyle 293 Expression Medium and Opti-MEM Reduced Serum Media were purchased from ThermoFisher (Walthma, MA). The oxidized lipids, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzePC) and 1-hydroxy-2-azelaoyl-sn-glycero-3-phosphocholine (2-Aze-LPC) were purchased from Avanti Polar Lipids (Alabaster, AL).
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8

Cell Culture Protocols for HEK293-F, HEK293-T, SCC47, and U87MG

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Suspension-adapted FreeStyle™ human embryonic kidney HEK293-F cells (American Type Culture Collection Manassas, VA, USA) were routinely cultured in GIBCO FreeStyle™ 293 expression medium (Thermo Fisher Scientific) at densities of 0.1 × 106 to 3.0 × 106 cells/ml )more than 90% of the cells were viable) in disposable Erlenmeyer tissue culture flasks with vented caps (TriForest, Irvine, CA, USA) at 125–135 rpm on an orbital shaker incubator (37 °C, 8% CO2). Cell density and viability were determined using a Countess II FL Cell Viability Analyzer (Thermo Fisher Scientific) according to the manufacturer's protocols.
HEK 293-T cells [American Type Culture Collection (ATCC), Manassas, VA, USA] and head and neck SCC4738 (link) cells (kindly supplied by Reidar Grénman, Department of Otorhinolaryngology—Head & Neck Surgery, Turku University, Finland) were grown in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Haemek, Israel). Glioblastoma U87MG cells (ATCC) were grown in MEM medium (Biological Industries). These two media were supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin (Biological Industries), referred to below as "plus supplements". The cells were placed in an incubator at 37 °C and 5% CO2.
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9

Cultivation of Mammalian Cell Lines for Viral Assays

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Female mammalian cells (FreeStyle™ 293-F cells, Thermo Fisher Scientific; HEK 293S, GnT I-/- cells, ATCC) were cultured in suspension in GIBCO™ FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) at 37°C in a Multitron Pro Shaker (Infors HT) with 70% humidity, 8% CO2 and rotating at 130 rpm. B lymphoblastoid BJAB cells (DSMZ)77 (link) were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Thermo Scientific) in a 37°C, 5% CO2 incubator. For pseudovirus neutralization assays, HEK293T cells (ATCC) and HEK293T-ACE2 cells (BEI NR52511) were cultured in DMEM media supplemented with 10% heat-inactivated FBS (Gibco), 2.5% HEPES (Gibco) and 0.5% gentamicin (ThermoFisher) in a 37°C, 5% CO2 incubator. Vero E6 cells (ATCC) were maintained in a humidified incubator at 37°C, 5% CO2 in DMEM (Corning) supplemented with 10% FBS.
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10

Detailed Protocol for Protein Expression and Purification

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All plasmids and primers used in this study are summarized in Supplementary Tables 1 and 2, respectively. All restriction endonucleases, T4 DNA ligase, and Vent polymerase were purchased from New England Biolabs (Ipswich, MA, USA). The Taq polymerase and oligonucleotide primers were from Biosesang (Seongnam, Republic of Korea) and Cosmogenetech (Seoul, Republic of Korea), respectively. DifcoTM Terrific Broth, Ni-NTA agarose, Protein A agarose, and glutathione agarose 4B were obtained from Becton Dickinson Diagnostic Systems (Sparks, MD, USA), Qiagen (Hilden, Germany), Genscript (Scotch Plains, NJ, USA), and Incospharm (Daejeon, Republic of Korea), respectively. An Alexa Fluor 488 labeling kit, 1-Step Ultra-TMB substrate solution, GIBCO FreeStyle™ 293 expression medium, and sheep anti-hC1q-HRP conjugate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyethyleneimine-Max and goat anti-GST-HRP conjugate were obtained from Polysciences (Taipei, Taiwan) and GE Healthcare (Piscataway, NJ, USA), respectively. Unless stated otherwise, all other biochemical reagents were purchased from Sigma‒Aldrich (St. Louis, MO, USA).
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