HEK293 and U2OS cells were cultured in Dulbecco's-modified Eagle's medium, containing 4.5 g/l D-glucose and GlutaMAX (Life Technologies, Carlsbad, CA, USA), and supplemented with 10% fetal bovine serum. HEK293 cell culture medium also contained pyruvate. A FLAG-DNA-PKcs-expressing stable cell line was generated by transfecting a mammalian expression vector encoding FLAG-DNA-PKcs fusion protein (gift from Dr. David J. Chen, UTSouthwestern, Dallas, TX, USA) with pTKHyg plasmid into 293H cells (Life Technologies) and selecting individual clones with 200 μg/ml of hygromycin B (hygB). Individual hygB-resistant clones were screened for full-length FLAG-DNA-PKcs expression by western blotting. One clone expressing physiological levels of tagged-DNA-PKcs was selected for further experiments. Suspension-adapted HEK293 (HEK293S) cells were cultured in Freestyle 293 medium (Life Technologies) supplemented with hygB (100 μg/ml) between densities of 0.5–3 × 106 cells per ml in conical flasks on a shaking platform (160 r.p.m.) in a humidified 37 °C incubator. HEK293S cells were transfected with pCMX-FLAG-XLS or pCMX-LacZ plasmid (gift from Dr. Thomas Perlmann, LICR, Stockholm, Sweden) using Freestyle MAX (Life Technologies) according to the manufacturer's instructions.
Freestyle max
Manufactured by Thermo Fisher Scientific
Sourced in Sweden, United States
The Freestyle MAX is a laboratory equipment product designed for automated sample preparation and processing. It is a modular and configurable system that can be customized to meet the specific needs of various applications. The core function of the Freestyle MAX is to provide efficient and reliable sample handling and processing capabilities in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
293h cells, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hyclone dmem, by GE Healthcare (1 mentions)
Padvantage vector, by Promega (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Sonifier 250, by Emerson (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose resin, by GE Healthcare (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
293fectin, by Thermo Fisher Scientific (1 mentions)
Pef bos, by Thermo Fisher Scientific (1 mentions)
Pgex 2t vector, by GE Healthcare (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
11 protocols using freestyle max
1
Stable Cell Line Generation for DNA-PKcs
2
Generating Stable Cell Lines for DNA Repair Proteins
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HEK293F cells were transfected using Freestyle MAX (Life Technologies) according to the manufacturer’s instructions or PEI (1.5 μg/ml). Stable HEK293 cell lines expressing FLAG-tagged PAXX, -XLF, and -XRCC4 were generated by co-transfecting mammalian expression vector encoding N-terminal FLAG-tagged proteins with pTKHyg plasmid into 293 H cells (Life Technologies) and selecting individual clones with hygromycin B (0.2 mg/ml). Individual hygromycin B-resistant clones were screened for expression of full length FLAG-tagged proteins by immunoblotting. A stable clonal HEK293 cell line expressing FLAG-tagged DNA-PKcs was used as described10 . HEK293F cells were cultured in FreestyleTM 293 medium supplemented with hygromycin B (0.1 mg/ml) between densities of (0.5–3) × 106 cells/ml in conical flasks on a shaking platform (160 rpm) in a humidified 37 °C incubator. RPE-1 cells were transiently transfected using Lipofectamine 2000 according to the manufacturer’s instructions (Life Technologies). Stable U2OS cell lines expressing N-terminal tagged EGFP- or mCherry-Pol λ or N-terminal tagged EGFP-FLAG-Ku70 fusion proteins were generated by transfection with PEI (2 μg/ml).
3
Antibody Production in CHO-S Cells
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Expression vector encoding antibody heavy chain and light chain of BR105 was transfected into CHO-S cells using freestyle MAX (Gibco), and the monoclonal stable cell line was generated by MTX selection and limiting dilution. For BR105 production, cells were cultured in 300 L bioreactors for 14 days in a fed-batch mode, and BR105 was purified using protein A affinity chromatography and ion exchange chromatography. 18D5,31 KWAR23,32 (link) 1H933 and SIRP434 on a human IgG1 backbone with N297A amino acid substitution in the Fc region backbone were expressed by Biointron Biological. Human IgG1 control, human IgG4 control, mouse IgG1 control and Hu5F9 were purchased from Biointron Biological. B6H12 was from BioXcell. Anti-human CD20 antibody (zuberitamab) and anti-HER2 antibody (Herceptin biosimilar, HS022) were produced in-house.
4
Site-Specific Etanercept Expression
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Cell density was adjusted to 1 × 106 cells/mL for transfection using FreeStyle™ MAX (Gibco™, Waltham, MA, USA, 16447100). The ratio of pU6-MCS-Cas9-EGFP-Puro-sgRNA and pCG1.1-HA-etanercept-EGFP was 1:1 for site-specific integration transfection, and only the pCG1.1-etanercept-EGFP plasmid was used for random integration. After 24 h of transfection, the cell density was adjusted to 5 × 105 cells/mL in T25 flask incubated at 37.0 °C and 7% CO2.
5
Transient Cell Transfection Protocol
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For assays with transiently-transfected cells, cells were generated by bulk transfection. At the time of transfection, 293-F or 293F.GCaMP6s.blast cells were diluted to 1 million/mL with FreeStyle-293 medium. Transfection was performed by combining equal amounts of pAdvantage vector (Promega Corp., Madison WI) and target DNA. Total DNA was 50 µg per 40 mL transfection. The transfection reagent was Freestyle MAX (Invitrogen). Cell viability at 24 hours was above 80% for transfections to be considered successful. Cells were spun down and resuspended in HyClone™ DMEM (GE Healthcare, Little Chalfont, UK) supplemented with 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA), then seeded into 384-well poly-D-lysine-coated plates at 10–15k cells/well at 16–24 hours after transfection, and used for assays 24–48 hours after transfection.
6
Antibody Expression in 293F Cells
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Antibody heavy- and light-chain variable regions were synthesized as “gBlocks” by Integrated DNA Technologies (www.idtdna.com ) and subsequently cloned into IgG and IgK expression vectors as previously described56 (link). Equal ratios of heavy- and light-chain plasmids were co-transfected into 293F cells using FreeStyle MAX (Invitrogen) according to the manufacturer’s instructions. Protein G columns were used to purify IgG as previously described57 (link).
7
Rapid Purification of His-tagged Proteins
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HEK293-F-FreeStyle cells (Invitrogen, Grand Island, NY) were transfected with plasmids encoding His, His-Si4 or Si4 fusion proteins using the Freestyle MAX reagent (Invitrogen, Grand Island, NY) and incubated for 24–72 hrs in 8% CO2. Cells were harvested and lysed by sonication (Sonifier 250, Branson, Danbury, CT). His-tag or His-Si4 fusion proteins were purified on Ni/NTA beads as previously described [30 (link)], with the exception that the Si4 tag was used to immobilize PK on silica NPs directly from the cell lysate (S1D and S1E Fig ), providing a fast and simpler purification process.
8
CHO-S and A20 Cell Transfection
9
Fc Fragment as Stradomer Unit
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Example 4
The Fc fragment has been used as a positive control in experiments described above in which the protein is coated and thereby fixed to plastic thereby exhibiting biological behavior that mimics coated IVIG. The Fc fragment also can be used as a core stradomer unit such as when it is attached to core moieties such as a liposome, a bead, or albumin. Further, we have demonstrated that the Fc fragment when cultured in certain expression systems and certain cell types, such as the Invitrogen FreestyleMax transient transfection system using CHO-S cells, can form higher order multimers on protein analysis, exhibit bivalent binding pattern on plasmon resonance imaging, and exhibit profound biological activity in soluble form comparable to coated IVIG in the immature DC assay. We conclude therefore that under certain carefully controlled conditions, the Fc fragment forms an Fc fragment stradomer. This effect may be due to post-translational modifications such as glycosylation changes.
10
Clec9A and RNF41 Protein Expression and Purification
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Clec9A ecto-domains and control proteins fused to a FLAG-tag and a biotinylation consensus sequence were expressed in mammalian Freestyle 293 F cells (Thermo Fisher) using Freestyle Max (Thermo Fisher) and purified using anti-FLAG M2 affinity gel (Sigma-Aldrich) and size exclusion chromatography (SEC) as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length FLAG-tagged Clec9A were subcloned into either pEF-Bos or pcDNA3.1+ (Invitrogen) for mammalian expression as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length and truncated versions of mouse and human RNF41 for mammalian expression (RNF41 full-length: 1-MGYD…VEEI-317; RNF41- ΔRING: 72-MRNM…VEEI-317) were subcloned into pcDNA3.1+ (Invitrogen). cDNA constructs encoding RNF41 for bacterial expression (RNF41-RBCC: 1-MGYD…VEEI-317; RNF41-BCC: 72-MRNM…VEEI-317; RNF41-CC: 135-IKHL…VEEI-317; RNF41-C: 169-DIQL…VEEI-317; RNF41-RBC: 1-MGYD…RAIR-181) were cloned into a modified pGEX-2T vector (GE Healthcare). His-tagged and untagged RNF41 proteins were expressed in mammalian Freestyle 293 F cells using 293Fectin (Thermo Fisher). GST-fusion RNF41 recombinant proteins were expressed in bacterial BL21 DE3 E. coli (Promega) and purified using Glutathione-Sepharose resin (GE Healthcare) and SEC.
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