Biology Reference
In-Depth Information
4.
5 mL of 0.25% trypsin-EDTA solution is added to each plate followed by
gentle swirling to evenly coat cells.
5.
The trypsin-EDTA solution is removed by aspiration and the cells are
placed in the tissue culture incubator for 3-5 min.
6.
10-15 mL of DMEM/10% FBS is added into each plate. Cells are dispersed
by gentle pipetting and then transferred to a 50-mL conical tube. The
volume is adjusted to
40 mL. After further mixing by inversion, the cells
are then split (3 mL/well) into the six-well plates precoated with collagen.
Gentle shaking is applied to distribute the cells evenly.
Note
: One 15-cm plate of adipocytes can be divided into 12 wells of 2 six-
well plates.
7.
After 2 days of standard incubation at 37
C, cells are washed twice with
room temperature DPBS.
8.
2.0 mL serum-free DMEM containing 5
m
g/mL of polybrene and an
10
8
pfu) is added into each well.
Note
: Please ensure that no serum is present since it may reduce the infection
efficiency of the virus. Moreover, we recommend varying the titer of virus
between 5
appropriate amount of adenovirus (2
10
8
pfu/well during the initial tries, as this
parameter can differ substantially between different genes.
9.
The plates are sealed with parafilm, placed securely in microplate adaptors,
and centrifuged for 1 h at 800
10
7
and 2
g
at room temperature.
Note
: Make sure
to tightly seal each plate with parafilm to prevent the entrance of bacteria
and other possible pathogens as well as the overspill of virus. Be sure to
set the deceleration speed of centrifuge to its minimum value. In order to
ensure the same centrifugal force of each well in the plate, following 30 min
of centrifugation the plate can be inverted for another 30 min of
centrifugation.
10.
After centrifugation, parafilm is removed and the plates are placed in the
tissue culture incubator.
11.
After 4 h, 2.0 mL of DMEM/10% FBS is added to each well in a step to aid
cell recovery.
Note
: Do not remove the virus-containing media at this point.
12.
After an overnight incubation, the virus-containing media is removed and
replaced with fresh DMEM/10% FBS. This media change is essential to
ensure the health of the cells as adenovirus and polybrene both have an overall
negative impact on cell viability during/following long-term incubation.
13.
After 2 more days of incubation, the cells are ready for functional analyses.
While the given time of 3 days is typical for efficient expression of
adenovirus-encoded proteins, anywhere in the range of 2-4 days following
infection could be ideal depending on the particular gene of interest.
3.
Combinational siRNA knockdown and adenoviral overexpression
1.
Adipocytes are transfected with siRNA by electroporation and plated in six-well
plates as described in “siRNA knockdown via electroporation” of
Section 2.2
.
2.
After 1 day following siRNA transfection, cells are washed twice with DPBS
and adenoviral infection is performed as detailed in “Adenovirus-mediated
overexpression assisted by centrifugation” of
Section 2.2
.
