Biology Reference
In-Depth Information
Dexamethasone stock solution
20
C)
10 mM in 100% ethanol (store at
Differentiation Media I
DMEM/10% FBS
1:500 IBMX stock solution
1:1000 insulin stock solution
1:10,000 dexamethasone stock solution
Differentiation Media II
10% FBS/DMEM
1:1000 insulin stock solution
DMEM/2% BSA
2% FA-free BSA in DMEM without phenol red
DMEM/0.5% BSA
0.5% FA-free BSA in DMEM without phenol red
Polybrene stock solution
10 mg/mL in water
Sterilize through a sterile 0.22-
m
m filter
Stealth siRNA solution
20
C)
100
m
M in sterile DEPC-treated water (aliquots stored at
6.2
METHODS
6.2.1
Adipocyte culture and differentiation
3T3-L1 preadipocyte cells have a fibroblast-like morphology and differentiate into
an adipocyte-like phenotype under appropriate conditions. Differentiated cells are
sensitive to lipogenic and lipolytic hormones and drugs, including epinephrine, iso-
proterenol, and insulin. For culturing and differentiating 3T3-L1 preadipocytes, we
have adapted a protocol originally developed by
Rubin, Lai, and Rosen (1977)
.
1.
Preadipocyte maintenance and passage
Mouse 3T3-L1 preadipocytes are maintained in DMEM/10% NCS in a
humidified atmosphere containing 10% CO
2
at 37
C. They are generally split at
a ratio from 1:5 to 1:10 depending on need, using 0.05% trypsin-EDTA. During
passage preadipocytes should be plated evenly and maintained at low density
(i.e., less than 70% confluence) at all times unless induction of differentiation is
planned. Spontaneous and partial differentiation would be triggered in
preadipocytes when high confluence is reached. If grown past 70% confluence
and subsequently passaged, preadipocytes that have experienced partial
differentiation will lose their ability to fully differentiate following hormonal
stimulation. Moreover, most cell lines are maintained in 5% CO
2
atmosphere.
However, 10% CO
2
appears to enhance the differentiation efficiency of
preadipocytes in comparison to 5% CO
2
.
