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2. Induction of adipogenic differentiation
1. Prior to differentiation, 3T3-L1 preadipocytes are routinely maintained in
15-cm plates with 25-30 mL of DMEM/10% NCS.
2. Upon reaching confluence, cells are replaced in fresh DMEM/10% NCS
and incubation is allowed to continue for 2 more days. This step further
promotes preadipocyte proliferation so that high cell density is achieved
to induce growth arrest and as a result improves the differentiation efficiency.
At the end of day 2, preadipocytes should appear tightly packed with
individual cells assuming narrow spindle-like morphology. Flat and spread
out cell shape often predicts a low efficiency differentiation after
hormonal induction.
3. To initiate differentiation, postconfluent preadipocytes are replaced in 25 mL
of Differentiation Media I (Day 0) freshly made from stock solutions of
different components. After thawing, insulin solution may be kept at 4 Cfor
up to 1 month. IBMX solution should be stored at
20 C continually,
which can be thawed and frozen up to three times. Since dexamethasone is
dissolved in ethanol, it will remain in liquid formeven at
20 C and is stable up
to1year.
4. Three days later (Day 3), the media become more viscous possibly
due to increased accumulation of FFAs and chondroitin sulfate proteoglycan-
I (versican) ( Okada et al., 2008 ) secreted by the cells. The media is
then carefully removed and changed to 25 mL of Differentiation Media II.
5. Two days later (Day 5), media is removed and cells are replaced in 25 mL of
normal DMEM/10% FBS.
6. Two to three days later (Day 7-Day 8), cells should be fully differentiated
and ready for subsequent functional experiments. For siRNA knockdown
or adenoviral overexpression experiments, at least 90% of the cells
present should be mature adipocytes with full and well-developed lipid
droplets. When plating cells into six-well plates after knockdown or
before virus infection, undifferentiated fibroblasts will quickly attach to the
six-well plate and begin proliferating prior to the attachment of mature
adipocytes, resulting in a large portion of the cells on the plates being
fibroblasts rather than adipocytes. In addition, usage of adipocytes that
have exceeded Day 8 of differentiation is not recommended, as they
are overly mature, do not easily attach to the plates, and are prone to
clumping.
6.2.2 Manipulation of gene expression
For knockdown of expression of particular genes, we routinely deliver specific
siRNA oligos into adipocytes by using an electroporation method adapted from a
plasmid transfection protocol ( Okada et al., 2003 ). For ectopic gene expression,
we have established an adenovirus-mediated method that is enhanced by polybrene
and centrifugation.
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