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]

Biosafety  classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

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 in this cell line. 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 IS 293-V (Irvine Scientific No. 91107). To make complete medium, add:

  • 200 mM L-Alanyl-L-Glutamine, 200 mM Solution in 0.85% NaCl (ATCC® No. 30-2115™) to a final concentration of 8 mM
  • 10% Poloxamer 188 (Sigma P5556) to a final concentration of 0.1%
  • 10uL/mL of ITS Solution (Insulin, Transferrin, Selenious Acid; ATCC® No. 30-2606™)
  • This medium is formulated for use with a 5-8% CO2 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. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count.

1. Seed at 5x105 cells/mL for a 2 day subculture and 4x105 cells/mL for a 3 day subculture (weekend)

2. To maintain high cell viability, prior to seeding, centrifuge cells for 5min at 170x g

3. Discard spent media and re-suspend cell pellet in pre-warmed fresh complete growth media

4.  Pipette cells gently to break aggregates

Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line. Alternately, appropriate amount of fresh media maybe added directly into the flask to adjust cell seeding density. However, cell viability might be slightly compromised and decreased by 5%.

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. GeneXPlus transfection reagent
    GeneX
    Date Updated: 3/27/2014
  2. 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
  3. 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
  4. 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
Restrictions

This material is available with the following restrictions: The cell line was derived from materials deposited at the ATCC by Rockefeller University and is provided for research purposes only. Neither the cell line nor the products derived from it may be sold or used for commercial purposes. Nor can the cells be distributed to third parties for purposes of sale, or producing for sale, cells or their products. The cells are provided as a service to the research community. They are provided without warranty of merchantability or fitness for a particular purpose or any other warranty, expressed or implied. Any proposed commercial use of the cells, or their products, must first be negotiated with Rockefeller University, Office of Technology Transfer, 1230 York Avenue, New York, NY 10065 Attn: Kathleen A. Denis, Associate Vice President Technology Transfer, Telephone: +1-212-327-8266, email: Kathleen.Denis@rockefeller.edu