Frequently Asked Questions
Note: For complete instructions on the use of any Gibco Cell Culture products, please refer to the "Product Instructions" link located on each product page. For additional technical support, please call 800-955-6288.
Using this nomenclature system, a continuous cell line is a population of cells that has undergone a genetic transformation, resulting in indefinite growth potential. Continuous cell lines are usually aneuploid. In practice, continuous cell lines can be cultured through a very high number of subcultures, although some further genotypic, and therefore phenotypic, changes may occur at very high passage numbers. Immortalization can occur spontaneously, or may be virally- or chemically- induced. It should be remembered that the working definitions of these terms can vary between research groups. Many researchers do not use the term “cell line” to refer to any population unless it has undergone a genetic transformation.
A cell strain is a subpopulation of a cell line that has been positively selected from the culture, by cloning or some other method. A cell strain will often have undergone additional genetic changes since the initiation of the parent line. Individual cell strains may, for example, have become more or less tumorigenic than the established line, or they may be designated as a separate strain following transfection procedures.
The term cell type refers to all cells with a common phenotype, eg keratinocyte, melanocyte. Therefore keratinocytes isolated from a number of different donors are all the same cell type.
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The term passage number refers to the number of times that a cell population has been removed from the culture vessel and undergone a subculture (passage) process, in order to keep the cells at a sufficiently low density to stimulate further growth. At Invitrogen™, the first culture following the isolation of cells from tissue is termed the primary culture. Following the first subculture, the cells are described as a secondary culture (or passage 1). After the second subculture, the cells become a tertiary culture (or passage 2), and so on.
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- Prepare a beaker of water at 37° C.
- Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes.
- Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
- Dip the lower half of the vial into the 37° C water to thaw.
- When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.
- Open the vial and pipette the suspension up and down with a 1 ml pipette to disperse the cells.
- Remove 20 ml from the vial and dilute the cell suspension in 20 ml of trypan blue solution (for example: Invitrogen™ Cat. # 15250-061).
- Use a hemacytometer to determine the number of viable cells per ml.
- Dilute the contents of the vial (1 ml) to the concentration recommended by the product instructions (for example 1.25 x 104 viable cells/ml for neonatal human epidermal keratinocytes from GIBCO® Cell Culture).
- Add 5 ml of cell suspension to each 25 cm² culture flask or 15 ml of cell suspension to each 75 cm² culture flask.
- Following inoculation, swirl the medium in the flasks to distribute the cells. Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns.
- Incubate the cultures in a 37° C, 5% CO²/95% air, humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.
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Please note: Due to differences in cryopreservation equipment and individual techniques, Invitrogen™ cannot guarantee that cells cryopreserved using this protocol will be viable upon recovery from cryopreservation, and does not provide a warranty for cells cryopreserved in an investigator's laboratory.
- Thaw Synth-a-Freeze® in a 37° C water bath or overnight at 4° C.
- If thawed in a water bath, do not exceed 37° C and do not leave the product at 37° C for an extended period of time.
- Synth-a-Freeze® should be equilibrated to 4° C prior to use. For optimal results, the use of a controlled-rate freezer is recommended. In the absence of a controlled-rate freezer, a cell cryopreservation container (such as Mr Frosty®, available from Nalge Nunc International) may be useful.
- If enzymatic agents are used to remove the cells from a culture surface, resuspend the cells in a solution that will neutralize the effects of the enzyme.
- Pellet the cells by centrifugation.
- After removing the supernatant, resuspend the cell pellet in cold Synth-a-Freeze® medium at a concentration of 5 x 105 to 3 x 106 cells/ml.
- Distribute the cell suspension in an appropriate number of cryopreservation vials.
- Cool the vials of cells to 4° C as quickly as possible.
- If using a controlled-rate freezer: Freeze the material by reducing the temperature 1° C per minute until the temperature reaches -40° C. Then reduce the temperature at a rate of 2° C per minute until the temperature reaches approximately -90° C.
- If using a cell cryopreservation container: Prepare the container according to the manufacturer’s instructions.
For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached -80°C.
As a substitute for Synth-a-Freeze®, the recommended basal medium for the cell type being cryopreserved, supplemented with 10% fetal bovine serum (FBS) and 10% DMSO, may be used. Please note that Synth-a-Freeze® is NOT recommended for the cryopreservation of human epidermal melanocytes. Synth-a-Freeze® is a trademark of Invitrogen™ Corp. Mr Frosty® is a trademark of Nalge Nunc International.
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Q: How can I calculate the correct rotor speed (RPM) to use for pelleting my cells by centrifugation?
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