||L1 is a continuous substrain of 3T3 (Swiss albino) developed through clonal isolation.
||This cell line is a suitable transfection host.
||The cells undergo a pre-adipose to adipose like conversion as they progress from a rapidly dividing to a confluent and contact inhibited state. A high serum content in the medium enhances fat accumulation [PubMed ID: 4426090].
||Dulbecco's Modified Eagle's Medium (DMEM) modified with 4500 mg/L glucose and bovine calf serum to a final concentration of 10%.
||NOTE: Never allow culture to become completely confluent.
Remove medium, and rinse with PBS without calcium and magnesium. Remove the solution and add an additional 1 to 2 mL of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37°C) until the cells detach.
Add fresh culture medium, aspirate and dispense into new culture flasks.
The recommended inoculum is 2 to 3 X 103 cells/cm2. Subculture before cultures become 70 to 80% confluent or when cells reach 5 to 6 X10(4) viable cells/cm2.
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
|Subculturing Medium Renewal
||2 to 3 times a week
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Growth Conditions: The serum used is important in culturing this line. Calf serum is recommended and not fetal bovine serum.
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
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. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. 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.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: 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 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).
||Green H, Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell 3: 127-133, 1974. PubMed: 4426090
Green H. Triglyceride-accumulating clonal cell line. US Patent 4,003,789 dated Jan 18 1977
Goodrum FD, et al. Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells. J. Virol. 70: 6323-6335, 1996. PubMed: 8709260
Scherer PE, et al. Identification, sequence, and expression of caveolin-2 defines a caveolin gene family. Proc. Natl. Acad. Sci. USA 93: 131-135, 1996. PubMed: 8552590
Kallen CB, Lazar MA. Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes. Proc. Natl. Acad. Sci. USA 93: 5793-5796, 1996. PubMed: 8650171
||Hugo Armelin - Universidade de Sao Paulo.
Aldo Angelo Moreira Lima - UNIVERSIDADE FEDERAL DO CEARÁ;
Marcelo Lima Ribeiro - Universidade São Francisco
Claudia Maria Oller do Nascimento