Biomedical Engineering Reference
In-Depth Information
cleaned with 70 % ethanol, and it is kept at room temperature
for an additional 5 min.
8. The cells are further processed as described in Subheading
3.4
.
1. The cells fi xed with 3 % paraformaldehyde are washed once
with 200
3.4
Staining of Cells
L/well of PBS at room temperature.
2. Residual paraformaldehyde is quenched by washing once with
200
ʼ
L/well 50 mM ammonium chloride solution and three
times with 200
ʼ
L/well of PBS at room temperature.
3. The cells are then permeabilized with 100
ʼ
L/well 0.1 % Triton
X-100 in PBS for 1 min at room temperature. 100
ʼ
L/well
PBS is added to the cells, and they are washed four times with
200
ʼ
L/well of PBS. Experiments not requiring antibody
staining should continue with
step 4
, experiments requiring
antibody staining should continue with
step 5
(
see
Note 6
).
4. The actin cytoskeleton and the nuclei are stained by the addi-
tion of 30
ʼ
L/well Alexa Fluor Phalloidin (fi nal concentration
1 U/mL) and Hoechst 33342 (fi nal concentration 10
ʼ
M) in
PBS, respectively, for 30 min at room temperature. The cells
are washed three times with 200
ʼ
ʼ
L/well of PBS at room tem-
L/well of PBS is left on the
plate, and it is stored protected from light at 4 °C until imaged.
Plates may be stored for at least 1 week before imaging.
5. In case antibody staining is required, 200
perature. After the last wash, 200
ʼ
L/well 1 % BSA in
PBS is added to the cells for 15 min at room temperature.
6. The primary antibody is diluted in 1 % BSA in PBS, and 30
ʼ
L
is added per well. The cells are incubated at room temperature
for 2 h. The appropriate antibody concentration has to be
determined for every antibody individually.
7. The cells are washed four times with 200
ʼ
ʼ
L 1 % BSA in PBS
per well.
8. The fl uorescent secondary antibody, Alexa Fluor Phalloidin
(fi nal concentration 1 U/mL), and Hoechst 33342 (fi nal con-
centration 10
L is
added per well. The cells are incubated at room temperature
for 30 min. The appropriate antibody concentration has to be
determined for every antibody individually.
9. The cells are washed four times with 200
ʼ
M) are diluted in 1 % BSA in PBS, and 30
ʼ
L/well of PBS at
room temperature, after which the plate is stored protected
from light at 4 °C until imaged.
ʼ
3.5 Automated
Image Acquisition
1. The bottom of the microplate is cleaned with 70 % ethanol and
loaded into the microscope. Before imaging, the parameters of
the microplate (number of rows and columns, well diameter,
column and row offset, column and row spacing, well depth,
