CHO INSR 1284
CRL-3307
Product categoryAnimal cells
OrganismCricetulus griseus, hamster, Chinese
TissueOvaryApplications3D cell culture
Product formatFrozenStorage conditionsVapor phase of liquid nitrogen
Specific applicationsThese cells can be used to investigate metabolic and mitogenic signaling of insulin metabolism and its metabolites. This cell line stably expresses the human insulin receptor B (M10051). Special Applications: Potency determination for human insulin and human insulin analogs such as insulin glargine [Lantus, (GlyA21,Arg B31,Arg B32) insulin].
Cells per vialApproximately 2.0 to 3.0 x 106Volume1.0 mLGrowth propertiesAdherentGenderFemaleStrainK1
CommentsFlp-In Cells (CHO-K1) cells were transfected with the gene for human insulin recepor B (M10051, NCBI GenBank), inserted in the basic vector pCDNA5-FRT-TO (cat.V6520-20 from Invitrogen)
Unpacking and storage instructionsCheck all containers for leakage or breakage.
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.
Complete mediumThe base medium for this cell line is Hams F-12 w/ Glutamax (Gibco 31765-035). To make the complete growth medium, add the following components to the base medium: 56 ml heat inactivated fetal bovine serum FBS and 3.4 ml 50 mg/mL Hygromycin B stock solutionTemperature37°C
Atmosphere95% Air, 5% CO2
Handling procedureTo 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 -70°C. Storage at -70°C will result in loss of viability.
Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
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.
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 7 minutes.
Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). 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). pH (7.0 to 7.6).
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 sheet.
Subculturing procedureVolumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
Remove and discard culture medium.
Briefly rinse the cell layer with Accutase to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of Accutase 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.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Cultures can be established between 2 x 104 and 4 x 104 viable cells/cm2.
Incubate cultures at 37°C.
Interval: MMaintain cultures at a cell concentration between 2 X 104 and 2.8 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Bacterial and fungal testingNot detectedViability≥ 50%
DepositorsS Ruggeber (Sanofi-Aventis Deutschland GmbH)Year of origin2008
