Lenti-X 293T Cell Line慢病毒包装细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

Lenti-X 293T Cell Line慢病毒包装细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心                      Lenti-X 293T Cell Line
Cat No.: 632180

The Lenti-X 293T Cell Line is a subclone of the transformed human embryonic kidney cell line, HEK 293, which is highly transfectable and supports high levels of viral protein expression (1). When transfected with the Lenti-X HTX Packaging System and a lentiviral vector, these cells are capable of producing lentiviral titers as high as >108 ifu/ml, as determined by flow cytometry. The cell line also constitutively expresses the simian virus 40 (SV40) large T antigen.
Package Contents
• 1 Flask Lenti-X 293T Cell Line (1.0~3.0 x 106 cells/Flask)
Storage Conditions
Store at 37oC
Shipping Conditions
RT
Product Documents
The following documents apply to this product:
• Lenti-X 293T Cell Line Protocol-at-a-Glance
Cell Type Information
The Lenti-X 293T Cell Line is a human embryonic kidney (HEK) cell line, transformed with adenovirus type 5 DNA, that also expresses the SV40 large T antigen. The cell line was subcloned for high transfectability and high-titer virus production.

Recommended Cell Culture Medium
90% Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (4.5 g/L), 4 mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10% Fetal Bovine Serum (FBS); 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. Add 1 mM sodium pyruvate (Sigma-Aldrich, S8636).

The flask was seeded with cells, grown, and completely filled with medium to prevent loss of cells during shipping.
1. Upon receipt, visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination. Also, check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable).
2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured.
3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 x g for 5 to 10 minutes. Remove shipping medium and save. Resuspend the pelleted cells in 10mL of this medium and add to 25 cm2 flask. Incubate at 37°C in a 5% CO2 in air atmosphere until cells are ready to be subcultured.

To insure 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 70°C. Storage at 70°C will result in loss of viability.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 10 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm2 or a 75 cm2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product.

Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
Subcultivation ratio: A subcultivation ratio of 1:4 is recommended. Medium renewal: Every 2 to 3 days.

Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the current publication of the Biosafety in Microbiological and Biomedical Laboratories from the U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes for Health.

Notes:
We recommend that cultures be initiated as soon as possible after receipt. For HEK 293-based cell lines, we recommend using collagen-coated plates or flasks for culturing for efficient recovery of frozen stocks. Culture vessels coated with compounds other than collagen may also provide suitable growth substrates for HEK 293-based cell lines; however, only collagen-coated plates (e. g. BD Falcon BIOCOAT Collagen I cellware) have been tested at Clontech. The cells may be cultured on regular flasks/dishes (e.g. noncoated flasks/dishes) after recovery; however, if adherence is poor, we recommend collagen-coated vessels for all culturing purposes. Complete attachment of newly thawed HEK 293-based cultures may require up to 48 hrs.
References
1. Pear, W.S., et al. (1993) Proc. Natl. Acad. Sci. USA 90(12):8392–8396.

Quality Control Data
Mycoplasma Contamination Test
This lot of cells has been tested and found to be free of Mycoplasma contamination.

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