Biomedical Engineering Reference
In-Depth Information
2. Incubate cells with virus suspended in fresh medium appropri-
ate for the target cells (
see
Note 8
). Allow infection to proceed
for the desired length of time.
3. Aspirate the virus-containing medium and wash infected cells
once with 2 mL of PBS. Add 100
L of 0.25 % trypsin-EDTA
and incubate at 37 °C for 5-10 min. Add 1 mL of medium to
the trypsinized cells and pipette the cell mixture up and down
several times to break up cell clumps. Transfer the resulting
single cell suspension to a 5 mL polystyrene tube.
4. Centrifuge the suspension for 5 min at 1,000 rpm. Aspirate the
supernatant and add 1 mL of cold 4 % formaldehyde to the
pelleted cells. Vortex briefl y and fi x for 15 min at room tem-
perature (
see
Note 9
). If fi xing with 100 % methanol, incubate
cells for 15 min on ice and skip
step 5
.
5. Pellet the cells, aspirate the supernatant, and add 1 mL of per-
meabilization buffer. Vortex briefl y and incubate for 15 min at
room temperature.
6. Pellet the cells, aspirate the supernatant, and add 1 mL of
blocking buffer. Vortex briefl y and incubate for 15 min at
room temperature.
7. Pellet the cells, aspirate the supernatant, and add 100
ʼ
L of
primary antibody diluted in blocking buffer. Incubate for 1 h
at room temperature or overnight at 4 °C (
see
Note 10
).
8. Wash the stained cells by fi rst centrifuging at 1,000 rpm, then
aspirating the antibody, and adding 1 mL of blocking buffer.
Vortex briefl y and repeat at least one more time.
9. Pellet the cells, aspirate the supernatant, and add 100
ʼ
L of
species-specifi c secondary antibody diluted in blocking buffer.
Ensure the secondary antibody is conjugated with appropriate
Alexa Fluor Dyes. Incubate for 30-60 min in the dark.
10. Wash the stained cells as above, then aspirate the antibody, and
add 1 mL of blocking buffer. Vortex briefl y and repeat at least
one more time.
11. Analyze the sample with a fl ow cytometer using a fl uorophore-
specifi c channel. Analyze cytometry data with commercial soft-
ware such as FlowJo or freeware such as WinMDI.
ʼ
4
Notes
1. Proximity ligation assay reagents are expensive and substantial
savings can be achieved by minimizing the surface area to be
stained. We use 12 mm diameter round glass coverslips, which
can be effi ciently covered with as little as 30
L of solution.
Proper humidifi cation must be maintained throughout the
staining procedure to prevent volume loss due to evaporation.
ʼ
