Biomedical Engineering Reference
In-Depth Information
5. 300
l of the siRNA/transfection mixture was distributed
evenly over six wells (i.e., 50
ʼ
l per well) of a solid black 96-well
cell culture plate ensuring to exclude the outermost wells of
the plate (
see
Note 6
).
6. 50
ʼ
l of the HeLa cell suspension prepared above in
Subheading
2.1
,
item 1
was gently seeded into each well and
mixed by pipetting to ensure equal cell distribution of the cells
with the siRNA/transfection mixture. The plates were incu-
bated at 37 °C in a CO
2
incubator (
see
Note 7
).
ʼ
1. 24 h following transfection, the cells were serum starved in SF
DMEM. To remove media, the plates were fl icked with
medium force over a sink and 200
3.2 HGF-Induced
MET Internalization
l of SF DMEM was then
added to each well. The media change was repeated once more
(
see
Note 8
).
2. Following an additional 24 h (48 h following transfection),
media were removed and replaced with 100
ʼ
l of SF DMEM
or SF DMEM supplemented with 100 ng/ml HGF prewarmed
to 37 °C to the appropriate wells.
3. Cells were incubated for 15 min at 37 °C in a water bath.
4. For studies using Dynasore, appropriate wells were pretreated
with 50
ʼ
ʼ
l of prewarmed SF DMEM containing 50
ʼ
M
Dynasore for 30 min at 37 °C (
see
Note 9
).
5. Media were removed by fl icking the plate over a sink, and then
100
l of prewarmed SF DMEM or SF DMEM supplemented
with 100 ng/ml HGF, with or without 50
ʼ
M Dynasore, was
added to the appropriate wells. Plates were incubated at 37 °C
for the required time (0-15 min).
ʼ
3.3 Cell ELISA
for MET Internalization
1. All immunostaining steps were performed in the absence of
detergent to ensure the measurement of surface-associated
receptor. All of the required solutions were prepared in advance.
2. Following HGF treatment, cells were fi xed with freshly pre-
pared 200
l of 4 % paraformaldehyde in PBS for 10 min at
room temperature.
3. Cells were washed with PBS three times for 10 min each at
room temperature and then incubated with 100
ʼ
g/
ml goat anti-human MET antibody in PBS containing 10 %
horse serum or 100
ʼ
l of 0.2
ʼ
g/ml of goat IgG in 10 % PBS
containing 10 % horse serum for 1.5 h at room temperature.
4. Cells were washed with PBS 3 × 10 min at room temperature
and then incubated with 100
ʼ
l of 0.2
ʼ
l of secondary HRP-labeled
bovine anti-goat IgG in 10 % (v/v) horse serum in PBS for 1 h.
ʼ
5. Secondary antibody solution was discarded and replaced by
5
g/ml DAPI in PBS and incubated for 10 min at room
temperature.
ʼ
