293T/17 SF [HEK 293T/17 SF] (ATCC® ACS-4500)

Organism: Homo sapiens, human  /  Tissue: Kidney  / 

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Organism Homo sapiens, human
Tissue Kidney
Morphology Lymphocyte-like; single cells to small aggregates
Culture Properties suspension
Biosafety Level 2
Applications This cell line is optimal for transient transfection and protein expression
Storage Conditions liquid nitrogen vapor phase
Derivation HEK293T/17 SF cells are a derivative of the 293T (293tsA1609neo) cell line (ATCC CRL-11268), adapted to serum-free medium and suspension.
Antigen Expression SV40 T antigen

The cells constitutively express the temperature-sensitive SV40 T antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This feature increases protein expression levels by permitting more plasmid copies to persist in the transiently transfected cells. Expression vectors containing the human cytomegalovirus (CMV) promoter have been shown to achieve high levels of protein expression in 293T/17 cell line.

Transient transfections can be performed at small and large scale. High transfection efficiencies and protein yields have been demonstrated with ProteinX Plus Transfection Reagent (ATCC ACS-4004), HEKPlus SFM (ATCC ACS-4002) and HEKPlus Boost (ATCC ACS-4003). ATCC recommends passaging thawed cells at least twice prior to transfection to ensure optimal viability. Prior to transfection (24 hours), seed cells at a density of 8 x 105 cells/mL.

Complete Growth Medium The base medium for this cell line is ATCC HEKPlus SFM, (ATCC ACS-4002). To make the complete growth medium, add 200 mM glutamine to a final concentration of 8 mM. This medium is formulated for use with a 5-8% CO2 in air atmosphere.
Subculturing Subculture cells at log phase (when cells are ready for passaging, i.e., every 2-3 days, and are approximately 2 x 106 cells/mL). Pre-warm fresh growth medium prior to use. Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line.
1. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count.
Cryopreservation Cells should be frozen at a high concentration (e.g., 5-7 x 106 cells/mL) and at a low passage number. The cells should be ≥ 85% viable prior to freezing.
1. Prepare 2X freezing medium (Complete Growth Medium supplemented with 15% DMSO) and store at 2°C to 8°C until ready to use.
2. Determine the viable number of cells and percent viability. Calculate the required volume of freezing medium based on the desired viable cell density per vial.
3. Centrifuge the cell suspension at 170 × g for 5 to 10 minutes. Carefully aspirate & discard supernatant.
4. Resuspend the cell pellet in Complete Growth Medium, and then add equal volume of the cold 2X freezing medium (prepared in step 1).
5. Transfer the cell suspension into cryovials (1 mL/vial). Continue to gently mix the cell suspension to avoid cell clumping and to keep the suspension at a homogeneous state.
6. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. If a controlled rate freezer is not available, an isopropanol freezing container also may be used (e.g., Mr. Frosty). Store cells at -80°C overnight. Follow manufacturer instructions for freezing cells in chambers.
7. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.
Population Doubling Time 24 hours
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