Biology Reference
In-Depth Information
Adipocytes
Preadipocytes
Adipogenic induction
Trypsinization + replating
Trypsinization
PBS wash
siRNA
C
Knockdown + overexpression
0.5 mL of PBS
24 h incubation
Replace in 1.5 mL/well of
DMEM containing adenovirus
Cell pellet
Replace in 1.5 mL/well of
DMEM containing adenovirus
Cuvette
1 h centrifugation
1 h centrifugation
Electroporation + replating
4 h incubation
4 h incubation
Add 1.5 mL/well of
DMEM/10% FBS
Add 1.5 mL/well of
DMEM/10% FBS
24 h incubation
20 h incubation
20 h incubation
Replace media
Replace media
Replace media
48 h incubation
24 h incubation
48 h incubation
A
B
Knockdown
Overexpression
Lipolysis
TG hydrolase activity
Western blot
FIGURE 6.1
Summary of the experimental procedures for (A) knockdown, (B) overexpression, or
(C) knockdown plus overexpression of genes of interest in fully differentiated 3T3-L1 adipocytes.
3. After 1 day of adenovirus treatment, the media is replaced by DMEM/10%
FBS.
4. After a total 3 days following siRNA transfection or 2 days following
adenovirus infection, cells are ready for functional studies ( Fig. 6.1 ).
6.2.3 Functional assays for lipolysis
To assay lipolysis, we typically measure both TG hydrolase activity in cell extracts
and FA or glycerol release from living cells. For TG hydrolase activity assay,
we have adapted an assay protocol published by Holm and Osterlund ( Okada
et al., 2003 ). Specifically, the TG hydrolase activity is measured in adipocyte
extracts by using emulsified triolein as substrate. Free oleic acid released during
the hydrolytic reaction is captured onto BSA, separated into the aqueous phase,
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