293T/17 SF [HEK 293T/17 SF]

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.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

The base medium for this cell line is BalanCD HEK293 (Irvine Scientific cat# 91165). To make the complete medium, add to 475 mL of the base medium:

• 20 mL of 200 mM L-glutamine (ATCC 30-2214) for a final concentration of 8 mM
• 5 mL of ITS (Corning cat# 25-800-CR) for a final concentration of 10 µL/mL

This medium is formulated for use with a 5-8% CO₂ air atmosphere.

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If, upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C will result in loss of viability.

1. Rapidly thaw cells by placing the cryovial in a 37°C water bath, swirling gently. Remove the cryovial from the water bath when only a few ice crystals are remaining.
2. Sterilize the cryovial by rinsing with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3.  Using a 1-mL or 5-mL pipette, transfer thawed cells drop-wise into 9 mL pre-warmed complete growth medium in a 15-mL centrifuge tube. Gently pipette the cells up and down several times to mix thoroughly.
4. Centrifuge the cell suspension at 170 × g for 5 minutes.
5. Carefully aspirate the supernatant and discard, leaving the cell pellet.
6. Gently resuspend the cell pellet in fresh pre-warmed complete growth medium, and transfer cell suspension into a filtered cap/non-baffled shaker flask. Cells should be seeded at a density of 5 x 10⁵ cells/mL.
7. Place the flask in a 37°C shaking incubator (125 to 130 rpm) with 5-8% CO₂. Note: Viability after initial thaw is generally lower; however, after 2-3 passages, the cells are fully recovered and reach optimal viability.

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.

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.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.