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Immortalized Old Mouse Brown Preadipocyte Cell Line

BHC10000002
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Contact us to order
Tel
+1 866.986.9598
Email
[email protected]
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Applications Range Study of energy homeostasis, thermogenesis, metabolic disorders, and aging processes
Shipping Shipped on dry ice
Product Format Frozen
Thawing Protocol 1. Take cryotubes containing preadipocytes out of a liquid nitrogen tank.
2. Keep cryotubes in a 37°C water bath for 2 to 3 min to thaw the cells.
3. Centrifuge cryotubes 3 min at 400 × g,4°C.
4. Discardsupernatant (freezing medium) and do not disturb the cell pellet in the bottom of the tube; the cell pellet could be visible or invisible.
5. Add 1mlcell growth medium into each cryotube, and gently vortex for 10 s.
6. Transfer the 1 ml cell suspension to a new 50-ml tube.
7. Add additional cell growth medium and gently vortex for 10 s.
8. Plate the cells in tissue culture plates, dishes, or flasks. To yield 40% to 50% confluence, seed 1 × 105 cells into one well of 6-well plate; 1 × 106 cells into one 10-cm dish; 1.5 × 106 cells into one T-75 flask.
9. Culture the cells in growth medium at 37°C in a humidified incubator with 5% CO2. 
Freezing Protocol 1. Wash preadipocytes with 1× PBS for one time and add a couple of drops of trypsin to cover the cell surface.
2. Incubate the cells at 37°C in the incubator for 2 to 3 min.
3. Add growth medium to the well (1 ml/well, 6-well plate).
4. Detach and mix cells by pipetting up and down.
5. Collect cells in a new 50-ml tube.
6. Centrifuge the cells 3 min at 400 × g,4°C.
7. Carefully remove the supernatant and avoid disturbing the cell pellet.
8. Add cell freezing medium to the tube and vortex for 30 s to resuspend cells. Usually, 3 ml freezing medium is used for the cells collected from one plate (6-well plate).
9. Aliquot 1 ml cells in a 1 ml cryotube.
10. Place cell cryotubes in a freezing container containing 100% isopropyl alcohol and keep them in a–80°C freezer overnight.
11. Store the cells in liquid nitrogen for long-term preservation.
Subculture Protocol When the preadipocytes reach 80% to 90% confluence, subculture cells with a ratio of 1:3 to 1:4, which will yield 30% to 40% confluence.

Materials
Preadipocytes (see Support Protocol 2 or Basic Protocols 3, 4, and 5)
1×PBS, calcium- and magnesium-free (Fisher, cat. no. 10-010-049)
0.25% trypsin-EDTA (1×) (Gibco, cat. no. 25200-056)
Primary preadipocyte growth medium (see recipe)
Cell culture incubator, 37°C, 5% CO2
Inverted microscope
50-ml Falcon tubes (Corning, cat. no. 352070)
Tabletop centrifuge
Vortexer
Tissue culture plates, dishes, or flasks

Procedures: 
 1. Aspirate culture medium, wash cells once with 1× PBS.
 2. Add 0.25% (w/v) trypsin-EDTA to cover the cell surface.
 3. Incubate the cells in an incubator at 37°C for 2 to 3 min.
 4. Observe cells under an inverted microscopy with 4× or 10× magnification. When fibroblast-shaped preadipocytes shrink and become dot-shaped cells (Fig. 2), add growth medium to inactivate trypsin (1 ml/well of a 6-well plate).
 5. Detach cells by pipetting up and down, and then collect cells to a new 50-ml tube.
 6. Centrifuge the cell suspension 3 min at 400 × g, room temperature.
 7. Carefully discard the supernatant (trypsin and medium), do not disturb the cell pellet in the bottom of the tube. The cell pellet may be visible or invisible.
 8. Add growth medium to the tube, and vortex for 10 s. Usually one 6-well plate of preadipocytes can split into three to four 6-well plates; add growth medium accordingly.
 9. Plate the cells in new tissue culture plates, dishes, or flasks.
 10. Culture the cells in the incubator at 37°C, with 5% CO2, to allow the cells to grow.
 11. Replace with fresh growth medium every other day. When the cells reach 80% to 90% confluence, either subculture cells again or freeze cells for cryopreservation.
SKU Supplier No. Your price Quantity
BHC10000002 102 -
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