FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37 °C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD [32 (link)] using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
Freestyle 293f medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
FreeStyle 293F medium is a serum-free, protein-free medium designed for the growth and transfection of HEK 293F cells. It provides a defined, animal-component-free environment to support the growth and recombinant protein production of HEK 293F cells in suspension culture.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 f cells, by Thermo Fisher Scientific (23 mentions)
Expifectamine 293 transfection reagent, by Thermo Fisher Scientific (19 mentions)
0.22 μm filter, by Thermo Fisher Scientific (15 mentions)
Nickel affinity column, by Thermo Fisher Scientific (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Protein g sepharose column, by GE Healthcare (3 mentions)
Protein a affinity column, by Cytiva (2 mentions)
0.22 m filter, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ni nta column, by Thermo Fisher Scientific (2 mentions)
Superose 6 10 300 column, by GE Healthcare (2 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Wisent (1 mentions)
Dulbecco modified eagle medium (dmem), by Wisent (1 mentions)
Ni nta resin, by GE Healthcare (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Bira500 kit, by Avidity (1 mentions)
Captiva primab, by Repligen (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
30 protocols using freestyle 293f medium
1
SARS-CoV-2 Spike RBD Protein Production
2
Maintenance of SARS-CoV-2 Spike Expressing Cell Lines
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The 293T human embryonic kidney (obtained from ATCC) and 293T-ACE2 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent) supplemented with 5% fetal bovine serum (FBS) (VWR) and 100 μg/mL of penicillin–streptomycin (Wisent). CEM.NKr CCR5 + cells (NIH AIDS reagent program) and CEM.NKr CCR5 + cells stably expressing the SARS-CoV-2 Spike glycoproteins (D614G, BA.1 or BA.4/5) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO) containing 10% FBS and 100 μg/mL of penicillin–streptomycin. The 293T-ACE2 and CEM.NKr CCR5+ cells stably expressing the SARS-CoV-2 Spike glycoproteins were previously reported [31 (link),34 (link),35 (link)]. The 293T, 293T-ACE2, and CEM.NKr CCR5 + cell lines were maintained at 37 °C under 5% CO2. FreeStyle 293 F cells were grown in FreeStyle 293F medium (Invitrogen) to a density of 106 cells/mL at 37 °C with 8% CO2 under regular agitation (135 rpm).
3
Transient Co-Transfection and Purification of Antibody and pMHC Proteins
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Plasmids encoding the heavy chain and light chain of Abs were transiently co-transfected in pairs, at equivalent molar ratios, into cultured mammalian human embryonic kidney HEK293F cells in Freestyle 293F medium (Invitrogen, CA, USA, 12338018) following the standard protocol [34 (link),35 (link)]. Culture supernatants were collected after 6 days by centrifugation and filtration (0.22 μm, polyethersulfone; Corning). Abs were purified from the culture supernatants using a CaptivA™ Protein A-agarose chromatographic column (Repligen, MA, USA) and were extensively dialyzed to achieve the final composition of phosphate-buffered saline (PBS; pH 7.4). Likewise, the plasmid encoding the pMHC SCT protein was transfected into HEK293F cells. The pMHC protein was purified from the culture supernatant using Ni-NTA resin (GE Healthcare, IL, USA). Protein concentrations were determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA). To prepare an Ab-screening antigen, the purified pMHC SCT proteins were biotinylated using a BirA500 kit (Avidity LLC, Colorado, USA) following the manufacturer’s instructions [35 (link)].
4
Expression and Purification of EGFR Proteins
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All ER-PE24 ITs and anti-EGFR monobody affinity variants were solubly expressed at 20 °C for 48 h in E. coli strain SoluBL21(DE3) (Genlantis, San Diego, CA, USA) [15 (link)], purified using Ni-NTA resin (Qiagen, Hilden, Germany; 30210), and finally formulated in a phosphate-buffered saline (PBS) buffer (2.67 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4, pH 7.4) [34 (link)].
For the expression of EGFR-ECD-Fc protein, the corresponding plasmid was transiently transfected into HEK293F cell cultures in Freestyle 293F medium (Invitrogen, Waltham, MA, USA) according to the standard protocol [35 (link)]. Human EGFR-ECD-Fc protein was purified from the culture supernatants using protein-A agarose resin (Captiva PriMAB, Repligen, Waltham, MA, USA) and finally formulated in a PBS buffer. The concentration of purified proteins was determined using the Bicinchoninic Acid (BCA) assay.
For the expression of EGFR-ECD-Fc protein, the corresponding plasmid was transiently transfected into HEK293F cell cultures in Freestyle 293F medium (Invitrogen, Waltham, MA, USA) according to the standard protocol [35 (link)]. Human EGFR-ECD-Fc protein was purified from the culture supernatants using protein-A agarose resin (Captiva PriMAB, Repligen, Waltham, MA, USA) and finally formulated in a PBS buffer. The concentration of purified proteins was determined using the Bicinchoninic Acid (BCA) assay.
5
Production of SARS-CoV-2 Spike RBD Protein
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FreeStyle 293 F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37 °C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDSPAGE gels and stained with Coomassie Blue.
6
Recombinant SARS-CoV-2 RBD Protein Production
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FreeStyle 293 F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 106 cells/mL at 37 °C with 8% CO2 under regular agitation (150 rpm). Cells were transfected with the plasmid coding for SARS-CoV-2 S RBD WT using an ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant RBD proteins were purified using nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
7
Recombinant ACE2 Protein Production
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FreeStyle 293F cells (Invitrogen, Rockford, IL, USA) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37 °C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for soluble ACE2 or ACE2-Fc using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 µm filter (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant sACE2 protein was purified by nickel affinity columns (Invitrogen) and ACE2-Fc was purified using protein A affinity column (Cytiva, Marlborough, MA, USA), as directed by the manufacturers. The protein preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80 °C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie blue.
8
Recombinant inRas37 Protein Production
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Based on our previous study, plasmids generating heavy chain (pciw3.4-inras37-HC) and light chain (pciw3.4-inRas37-LC) were produced. Escherichia coli-containing plasmids were cultured in Luria-Bertani medium on a small scale (3 mL) by incubating for 12 h at 37°C and 200 rpm, and then incubated on a large scale (1 L) for 12 h at 37°C and 200 rpm. After 12 h of incubation, E. coli were lysed and purified to obtain the plasmid DNA using a MN (Macherey-Nagel) kit (Düren, Germany), and the plasmid DNA products were transiently co-transfected in pairs at an equivalent molar ratio into 200 mL of HEK293F cells (2×106 cells/mL) in Freestyle 293F medium (Invitrogen, CA, USA). The transfected cells were cultured for 7 days in an incubator at 37°C and 125 rpm, and the cell supernatants were centrifuged at 3,000 rpm and filtered (0.22 μm, Polyethersulfone; Corning, NY, USA). inRas37 was purified from cell supernatant using a protein A-resin (Repligen, MA, USA) at a 1 mL/min flow rate and then dialyzed to achieve a final buffer composition of Histidine buffer (pH 7.4) using a sephadex G-25 desalting columns (GE Healthcare, Chicago, IL, USA). Then inRas37 in buffer was filtered using cellulose acetate membrane filters (0.22 μm, Corning), and its concentration was determined by the absorbance at 280 nm using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA).
9
Recombinant SARS-CoV-2 RBD Protein Production
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FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37°C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S WT RBD (Beaudoin-Bussières et al., 2020 (link)) using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
10
Recombinant SARS-CoV-2 RBD Protein Production
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FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37°C with 8% CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue.
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