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

Organism: Homo sapiens, human  /  Tissue: Kidney  / 

Permits and Restrictions

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Organism Homo sapiens, human
Tissue Kidney
Product Format frozen
Morphology Lymphocyte-like; single cells to small aggregates
Culture Properties suspension
Biosafety Level 2  [Cells contain adenovirus]
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
Comments

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
Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
FAQ's
  1. HEKPlus Protein Expression System Parameters
    Cell density at transfection, ratio of DNA to GeneX
    Date Updated: 3/27/2014
  2. GeneXPlus transfection reagent
    GeneX
    Date Updated: 3/27/2014
  3. HEKPlus Protein Expression System vectors
    Expression vectors containing the SV40 origin of replication should be used. These vectors permit more plasmid copies to persist in the transiently transfected cells.
    Date Updated: 3/27/2014
  4. HEKPlus SFM supplements
    Complete culture medium is prepared by adding L-glutamine to the HEK
    Date Updated: 3/27/2014
  5. HEKPlus Protein Expression System time for optimal protein expression
    It usually takes 2 to 3 days to see optimal expression of intracellular proteins and 6 days for optimal expression of secreted proteins. It is recommended that a time course be performed for...
    Date Updated: 3/27/2014
  6. Advantage of using HEKPlus Protein Expression System
    The HEK
    Date Updated: 3/27/2014
  7. HEKPlus Protein Expression System advantages
    The HEK
    Date Updated: 3/27/2014
  8. HEKPlus Protein Expression System to express hard-to-express proteins
    Yes. The HEK
    Date Updated: 3/27/2014
  9. HEKPlus Protein Expression System Scale
    The HEK
    Date Updated: 3/27/2014
  10. HEKPlus Protein Expression System secreted proteins
    It is recommended to collect cell supernatants at day 6 post-transfection. However, one may want to perform a time course study to determine optimal time for collection post-transfection for each...
    Date Updated: 3/27/2014
  11. HEK293T/17 SF cells are clumping
    No. Small aggregates are easily dispersed with minimal pipetting and do not affect the performance of the cell line.
    Date Updated: 3/27/2014
  12. Replenishing medium for HEK293T/17 SF cells
    Yes. Incomplete media changes affect the cell viability and recovery. Spent medium should be completely removed and replaced with fresh medium every 2 to 3 days.
    Date Updated: 3/27/2014
  13. Media volume for HEK293T/17 SF cells
    For protein expression, it is important to have adequate shaking in the culture vessel. We usually add culture medium to 20% volume of a shaker flask.
    Date Updated: 3/27/2014