HEK293 cell line was cultured in complete Dulbecco's modified Eagle's medium (Cytiva) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA). 293 A and 293 T cells were incubated at 37 °C in 5% CO2, and 293 F cells were grown in serum-free medium (SFM4HEK293; Cytiva) at 37 °C in a humidified 8% CO2 incubator rotating at 150 rpm.
Sfm4hek293
Manufactured by Cytiva
Sourced in United States
The SFM4HEK293 is a serum-free, animal component-free medium designed for the growth and maintenance of HEK293 cells in suspension culture. It is formulated to support the proliferation and viability of HEK293 cells without the need for animal-derived supplements.
Automatically generated - may contain errors
3 protocols using sfm4hek293
1
HEK293 Cell Culture Conditions
2
HEK 293SF Cell Culture and Adenoviral Transduction
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The HEK 293SF cell line was derived from HEK 293S cells, and adapted to grow in suspension and serum-free media through multiple steps of adaptation and clone selection [22 (link)]. Cells were grown in suspension in shake flasks (Corning) with an agitation of 120 rpm using orbital shakers (Infors HT, Anjou, QC, Canada), 5% CO2 at 37 °C. Cells were passaged regularly when reaching densities ~1 × 106 cells/mL.
The following culture media were utilized in this study: Pro293s-CDM (Lonza, Walkersville, MD, USA), SFM4Transfx-293 and SFM4HEK293 (both from Cytiva, Logan, UT, USA), Ex-Cell 293 (Sigma-Aldrich, St. Louis, MO, USA) and an in-house-developed serum-free medium (HEK SFM).
The adenovirus used for infection was an Ad5-containing green fluorescence protein (Ad5-GFP) under the control of the CMV promoter. Viral stock titer was 1.7 × 1010 IVP/mL and aliquots were stored at −80 °C for subsequent analysis.
The following culture media were utilized in this study: Pro293s-CDM (Lonza, Walkersville, MD, USA), SFM4Transfx-293 and SFM4HEK293 (both from Cytiva, Logan, UT, USA), Ex-Cell 293 (Sigma-Aldrich, St. Louis, MO, USA) and an in-house-developed serum-free medium (HEK SFM).
The adenovirus used for infection was an Ad5-containing green fluorescence protein (Ad5-GFP) under the control of the CMV promoter. Viral stock titer was 1.7 × 1010 IVP/mL and aliquots were stored at −80 °C for subsequent analysis.
3
Production and Purification of Recombinant CAR Protein
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The nucleotide sequence of the extracellular active region with a signal peptide of the coxsackie adenovirus receptor (CAR) was synthesized by Tsingke Biotechnology Co, Ltd. and cloned into a pCMV-mFc expression vector. Recombinant plasmid was transfected into 293 F cells grown in serum-free medium (SFM4HEK293; Cytiva, Marlborough, MA, USA) after 4 days of cell culture. The supernatant was collected by clarification filtration, and the protein was purified using AKTA Pure 25 with PrismA protein A and Superdax200 increase column (Cytiva). The specificity and purity of the CAR recombinante protein was examined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. S1 ). Protein concentrations were determined using a bicinchoninic acid assay (BCA) kit and the samples were stored at −80 °C.
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