Biology Reference
In-Depth Information
13.
Transfer the cell suspension from (11) into the tube containing siRNA and
mix by gentle pipetting three times using the P-1000 pipettor. The final
concentration of siRNA in the electroporation mixture is 5
M.
Note
:
siRNA concentration necessary for efficient knockdown is gene dependent.
We suggest varying siRNA doses from 2 to 10
m
M during the initial
tries. Additionally, a better knockdown effect may be achieved by mixing
siRNA oligos targeting different sequences in a gene of interest.
14.
The mixture of cells/siRNA is transferred into an electroporation cuvette.
Immediately place the cuvette into the Bio-Rad Gene Pulser electroporator
and deliver a single pulse at 950
m
F and 160 V.
15.
After pulse delivery, 1 mL of DMEM/10% FBS is added to the cells in
the cuvette. Immediate addition of the media is essential for readjusting the
pH following current delivery and ensuring continued cell viability.
16.
Transfer the electroporated cell suspension to a 15-mL conical tube
containing 11 mL of DMEM/10%, gently mix three times and incubate for
15 min at room temperature. This incubation period aids in both cell
recovery and separation of cellular debris (i.e., dead cells) that will float to
the surface of the suspension.
17.
Carefully remove the floating cell debris with a P-1000 pipettor.
18.
Split the cell suspension into four wells of a six-well plate by adding
3 mL/well. Distribute cells across the plate evenly by shaking gently and
then place in a tissue culture incubator.
19.
The following day, carefully change themedia and incubate for another 2 days.
Note
: If there appears to be a loss or lack of cell attachment, plates may be
precoated with collagen solution to promote adherence as described in (2) of
“Adenovirus-mediated overexpression assisted by centrifugation” in
Section 2.2
.
20.
Three days following electroporation, cells are ready for lipolysis assays.
While 3 days is typically good for maximal knockdown, genes do vary
slightly and it may be necessary to adjust this time frame according to the
particular gene of interest.
2.
Adenovirus-mediated overexpression assisted by centrifugation
1.
DPBS and DMEM/10% FBS are prewarmed to 37
C in water bath before
the experiment.
2.
Six-well plates are precoated with collagen solution. Make a 1:250 dilution
of collagen type I solution in DPBS and add 2.5 mL/well diluted collagen
solution to the plates. To allow establishment of a collagen base, plates are
placed in a tissue culture incubator for 1 h and then washed with DPBS
prior to the seeding of cells. It is an important step for the subsequent
centrifugation and virus infection protocols, since the presence of collagen
helps cells to remain adherent under stressful circumstances.
3.
Differentiated adipocytes (Day 7 or 8) in 15-cm plates are washed twice with
DPBS.
Note
: Again, differentiation efficiency needs to be at least 90% for
adipocytes to be useful.
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