Biology Reference
In-Depth Information
1.
siRNA knockdown via electroporation
1.
DPBS and DMEM/10% FBS media are prewarmed to 37
C in water bath
prior to conducting the experiment.
2.
Differentiated adipocytes (Day 7 or 8) in 15-cm plates are washed twice
with DPBS. The protocol below is optimized for one 15-cm plate of
adipocytes. Corresponding volumes of reagents or materials need to be used
for multiple plates of cells.
3.
5 mL of ice-cold 0.25% trypsin-EDTA solution is added followed by gentle
swirling to ensure even coating of the cells. We recommend using a high
concentration of trypsin (i.e., 0.25%) to detach adipocytes so that cell
clumping is minimized and a complete dispersion can be achieved.
4.
The trypsin-EDTA solution is removed by aspiration and the cells are
placed in the culture incubator for 5-10 min. To gauge the degree of
detachment, the cells are examined under a phase-contrast light microscope
every 2 min. Once detachment of the cells is complete, 10 mL of DMEM/
10% FBS is added to wash off the cells.
Note
: Tilt the plate and add the media to the top so that the cells flow to the
bottom of the plate. Make sure to NOT pipet cells, as this is a potential
avenue for cell damage.
5.
Cells along with the media are transferred to a 50-mL conical tube and more
DMEM/10% FBS is added to adjust the total volume to approximately
30 mL.
6.
Cells are centrifuged for 5 min at 200
g
at room temperature. To ensure the
formation of a firm pellet, the deceleration speed of the centrifuge should
be set to its minimum value (i.e., slowest speed).
7.
The supernatant is carefully removed by aspiration without disturbing the
cell pellet. After the addition of 30 mL of DPBS, the cell pellet is
resuspended by gentle inversion of the tube for five to six times.
8.
The tube is placed in a tube rack for 1-3 min at room temperature. The
clumped cells would settle to the bottom of the tube, which then can be
removed by gentle aspiration with a 5-mL pipette. Ensure to remove as many
of the cell clumps as possible, as they would create a nonuniform mixture
altering the dynamics of the cell suspension during electroporation.
9.
The cell suspension is centrifuged for another 5 min at 200
g
at room
temperature.
10.
Repeat steps (7)-(9).
11.
The final cell pellet is resuspended in 500
L of DPBS after supernatant
is removed. Use a P-1000 pipettor to mix the cells as gently and thoroughly
as possible. It is important to have a uniform suspension, ensuring even
current distribution during electroporation.
12.
In a separate tube, add 25
m
m
m
L of 100
M control or gene-specific siRNA
solution to the bottom.
Note
: Make sure to use RNase-free tubes to prevent possible degradation of
siRNA. We typically use a 5-mL polypropylene round-bottom tube fromBD
Falcon (#352063).
