HEK293T cells were kindly provided by Radu A. Aricescu32 (link) and were maintained as adherent monolayers in standard Dulbecco’s Modified Eagle’s Medium (DMEM, 4.5 g/l glucose, Institute of Molecular Genetics, The Czech Academy of Sciences, Prague) supplemented with 4 mM L-glutamine, non-essential amino acids and 10% foetal bovine serum (GIBCO Invitrogen, USA) in standard flasks (TPP, Switzerland) in a humidified 37 °C, 5% CO2 incubator. Suspension adapted HEK293T cells (0.25–6 × 106/ml) were maintained in EX-CELL 293 serum-free medium (Sigma, USA) supplemented with 4 mM L-glutamine in standard dishes (TPP, Switzerland) or square-shaped glass bottles with gas permeable caps (DURAN, Germany) using 30–40% of the nominal volume at 135 rpm (Orbit 1000 orbital shaker, rotational diameter 19 mm; Labnet, USA; bottles were fixed with Sticky Pad adhesive mat; New Brunswick Scientific, USA) placed within the same incubator45 (link).
Ex cell 293 serum free medium
Manufactured by Merck Group
Sourced in United States
EX-CELL 293 is a serum-free medium designed for the cultivation of HEK293 cells. It provides the necessary nutrients and growth factors to support the growth and expansion of HEK293 cells in a defined, animal component-free environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Procho5 medium, by Lonza (1 mentions)
Ni nta affinity column, by Qiagen (1 mentions)
Q sepharose fast flow, by GE Healthcare (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
Hitrap protein a hp column, by GE Healthcare (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
13 protocols using ex cell 293 serum free medium
1
Culturing HEK293T Cells: Adherent and Suspension
2
Optimized Cell Culture Conditions
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The human embryonic kidney (HEK) 293, HEK293T (ATCC) cell line and mouse NIH-3T3 cell (ATCC, Manassas, VA, USA) line were cultured in DMEM (Invitrogen, Waltham, MA, USA) supplemented with 10% FBS (BioWhittaker) at 37 °C in a 5% CO2 environment. ExpiCHO cells (Thermo Fisher, Waltham, MA, USA) were cultured in ProCHO5 medium (Lonza) and incubated with agitation at 31 °C and 4.5% CO2. HEK293E cells were cultured in an EX-CELL 293 serum-free medium (Sigma, St. Louis, MO, USA) and incubated with agitation at 37 °C and 4.5% CO2. Expi293F cells (Thermo Fisher, Waltham, MA, USA) were cultivated in an Expi293™ Expression Medium (Thermo Fisher, Waltham, MA, USA) at 37 °C and 8% CO2 on an orbital shaking platform with the shaking speed based on shaking diameter and flask size.
3
Transient Protein Expression in HEK293 Cells
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HEK293T cells were kindly provided by Radu A. Aricescu [24 (link)]. HEK293T and HEK293S GnTI− cells [25 (link)] (0.3–6 × 106/mL) were maintained in ExCELL293 serum-free medium (Sigma, St. Louis, MT, USA) supplemented with 4 mM L-glutamine in square-bottom glass DURAN flasks with gas permeable caps (DWK Life Sciences, Wertheim, Germany) using 30–40% of the nominal volume at 135 rpm on orbital shaker placed within the incubator at 37 °C and 5% CO2 [26 (link)]. Transient expression in both HEK293T and HEK293S GnTI− cell lines was performed using the plasmid pTW5sec, a derivative of the pTT5 plasmid backbone [41 (link)] modified in-house to contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a leader peptide of secreted alkaline phosphatase in frame with AgeI and KpnI cloning sites, followed by a C-terminal histidine tag. Therefore, proteins recombinantly expressed using this plasmid were secreted into the cell culture media and purified by immobilized metal affinity chromatography (IMAC).
4
Cell Culture Maintenance Protocols for DENV Research
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Vero E6 (Vero), HEp-2, and Huh7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum FBS; (Thermo Fisher Scientific) and 1% (vol/vol) penicillin-streptomycin (Thermo Fisher Scientific). Suspension-adapted HEK-DI-290-ORF cells were cultured in EX-CELL 293 Serum-Free Medium (Sigma-Aldrich) supplemented with 1% (vol/vol) penicillin-streptomycin, 1% (vol/vol) GlutaMAX-I (Thermo Fisher Scientific), and 50 nM PEP005 during DENV DIP production. All cells were incubated at 37°C in a humidified 5% CO2 atmosphere. C6/36 cells were grown in DMEM supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin at 28°C in a humidified 5% CO2 atmosphere.
5
Soluble αVβ6 Integrin Headpiece Purification
6
Cell Culture Maintenance Protocols for DENV Research
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Vero E6 (Vero), HEp-2, and Huh7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum FBS; (Thermo Fisher Scientific) and 1% (vol/vol) penicillin-streptomycin (Thermo Fisher Scientific). Suspension-adapted HEK-DI-290-ORF cells were cultured in EX-CELL 293 Serum-Free Medium (Sigma-Aldrich) supplemented with 1% (vol/vol) penicillin-streptomycin, 1% (vol/vol) GlutaMAX-I (Thermo Fisher Scientific), and 50 nM PEP005 during DENV DIP production. All cells were incubated at 37°C in a humidified 5% CO2 atmosphere. C6/36 cells were grown in DMEM supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin at 28°C in a humidified 5% CO2 atmosphere.
7
Generating Stable UBR5 Cell Lines
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The codon‐optimised sequence of full‐length human UBR5 was purchased from Twist Bioscience as DNA fragments and cloned into pMEPI plasmid as two constructs (UBR5 and UBR5wt). For single‐particle analysis, the UBR5 construct included a decahistidine (10×His) tag followed by a Prescission protease (3C) cleavage site, the codon‐optimised UBR5 sequence and a double strep tag. For all activity assays, the UBR5wt construct and its derivates included a double strep tag followed by a 3C site and the codon‐optimised UBR5 sequence. Mutations and deletions introduced to UBR5wt are listed in Appendix Table S1 . The constructs were used for the generation of stable HEK293T cell lines, generated by co‐transfecting the constructs under the control of a Tet inducible promoter bearing a puromycin selection marker and the PiggyBac transposase for stable genomic integration. Cells were cultured in FreeStyle 293 Expression Medium (GIBCO). Protein expression was induced by the addition of doxycycline at a final concentration of 1 μg/ml for 48 h. Twenty‐four hours post‐induction, 125 ml of Ex‐Cell 293 Serum‐Free Medium (Sigma Aldrich) was added per 0.5 l of culture. Cells were harvested by centrifugation at 2,000 g for 20 min. Pellets were washed in 1×PBS and flash‐frozen in liquid nitrogen for storage at −70°C.
8
Glycosylated UL7 Protein Expression and Antibody Production
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To express UL7 in a glycosylated form comparable to that of the native protein, stable clones were made by transfection of HEK293 with pCMV6-UL7Fc and selection with neomycin (G418; Gibco-BRL). Stable clones with G418 resistance were obtained by trypsinization of colonies, followed by plating at limiting dilutions onto a 96-well plate. Ten single clones were then expanded and analyzed for UL7 secretion by the use of mouse Fc and an IgG enzyme-linked immunosorbent assay (ELISA) kit (MednaBio). The clone producing the largest amounts of fusion protein was cultured in EX-Cell 293 serum-free medium (Sigma-Aldrich). The supernatant containing the fusion protein was purified using a HiTrap protein A HP column (GE Healthcare) according to the manufacturer’s specifications.
To generate an anti-UL7 serum, the recombinant protein was used for rabbit immunization. The serum obtained by bleeding at 10 days after the fourth immunization was purified on an HiTrap protein A HP column (GE Healthcare) according to the manufacturer’s specifications. An isotype control was generated by purifying rabbit serum prior immunization. Fc and CEACAM1 recombinant proteins were purchased at R&D Systems.
To generate an anti-UL7 serum, the recombinant protein was used for rabbit immunization. The serum obtained by bleeding at 10 days after the fourth immunization was purified on an HiTrap protein A HP column (GE Healthcare) according to the manufacturer’s specifications. An isotype control was generated by purifying rabbit serum prior immunization. Fc and CEACAM1 recombinant proteins were purchased at R&D Systems.
9
Cultivation of Human Cell Lines
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HEK293T cells were a kind gift from Prof. Radu A. Aricescu [66 (link)]. Cells were adapted for cultivation in suspension in EX-CELL 293 serum-free medium (Sigma, St. Louis, MO, USA) supplemented with 4 mM L-glutamine. Cells were maintained in the square-shaped glass bottles placed on an orbital shaker in a humidified incubator (37 °C, 5% CO2) [67 (link)].
HT-29 cells were provided by the Institute of Molecular Genetics, Czech Academy of Science, Prague. C33 cell line transfected with CAIX gene (C33_CAIX) or with a control vector containing neomycin resistance gene (C33_Neo) were kindly provided by Dr. Eliška Švastová (Institute of Virology, Slovak Academy of Sciences) [68 (link)]. Cells were cultivated in high glucose DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For splitting, cells were treated with trypsin/EDTA. Both C33 transfectants were maintained under 900 μg/mL of G418 antibiotics. If not mentioned otherwise, all cell-culture media and supplements were purchased from Sigma, St. Louis, MO, USA.
HT-29 cells were provided by the Institute of Molecular Genetics, Czech Academy of Science, Prague. C33 cell line transfected with CAIX gene (C33_CAIX) or with a control vector containing neomycin resistance gene (C33_Neo) were kindly provided by Dr. Eliška Švastová (Institute of Virology, Slovak Academy of Sciences) [68 (link)]. Cells were cultivated in high glucose DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For splitting, cells were treated with trypsin/EDTA. Both C33 transfectants were maintained under 900 μg/mL of G418 antibiotics. If not mentioned otherwise, all cell-culture media and supplements were purchased from Sigma, St. Louis, MO, USA.
10
Production and Purification of scFv-Fc Constructs
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The genes encoding the selected scFv clones were cloned into the pDR-OriP-Fc vector, which allows genetic fusion of scFv sequences to human gamma-1 Fc and hinges domain sequences for transient expression of scFv-Fc as described previously [23 (link)]. The constructed expression plasmid was transfected using polyethyleneimine (PEI, Polysciences, Warrington, PA, USA) into 293E (CRL-10852, ATCC) cells cultivated in suspension with Ex-Cell 293 serum-free medium (Sigma-Aldrich, St. Louis, MO, USA) at 37 ℃ in 8% CO2. The transfected cells were cultured for seven days at 32 ℃ in 8% CO2 while being fed with 15% glucose (Thermo Fisher Scientific, Waltham, MA, USA) and 200× Glutamax (Thermo Fisher Scientific, Waltham, MA, USA) twice. For purification of scFv-Fc, the supernatant was subjected to affinity chromatography based on a protein G-agarose column (Merck Millipore, Darmstadt, Germany).
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