D16

Brown fat generates heat via the mitochondrial uncoupling protein UCP1, defending against hypothermia and obesity. The isolation of ‘beige’ cells from murine white fat depots which resemble white fat cells show them to have extremely low basal expression of UCP1, but, like classical brown fat, they respond to cyclic AMP stimulation with high UCP1 expression and respiration rates and also beige cells have a gene expression pattern distinct from either white or brown fat and are preferentially sensitive to the polypeptide hormone irisin were produced with different clones. The CRL-3281 is one of the beige clones that, showing evidence that previously identified brown fat deposits in adult humans are composed of beige adipocytes, offers a foundation for studying this mammalian cell type with therapeutic potential for the epidemic of obesity and diabetes.

Primary stromal vascular fraction from inguinal depots of 7-9 week old 129SVE mice (Taconic) was fractioned and cultured as described (J Clin Invest 121(1):96-105, 2011.) Primary SVF cells were immortalized with 3T3 protocol as described (J Cell Biol 17:299-313, 1963.). Limiting dilution of cells into 96-well plates was used to derive subclones of parental immortalized SVF.

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.

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 O-ring 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 7 minutes.
4. 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).
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 sheet.

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.

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