FCM : Flow Cytometry
2. Immunofluorescent Staining for Flow Cytometry
a. Keep all the
incubations at 4 degree centigrade.
b. Harvest 2-6x10 cells and wash with PBS.
c. Incubate the cells with desired primary antibody concentration
at 50 ul PBS-FCS for 30 minutes in a 0.5 ml centrifuge tube. The antibody concentration should be optimized according
to the manufacturer's instruction and your own experience.
d. Centrifuge the tube at 1000 rpm for 3 minutes, and discard the supernatant.
Wash 3 times with PBS-NaN3, 5 minutes each.
e. Incubate with fluorescent conjugated second antibody in 50 ul PBS-FCS for 30 minutes.
Again, the concentration of #2 antibody should be optimized.
f. Centrifuge the tube at 1000 rpm for 3 minutes and discard the
supernatant. Wash 3 times with PBS-NaN3, 5 minutes each.
g. Fix the cells with 100 ul 4% paraformaldehyde for 10 minutes at room temperature.
h.
Centrifuge the tube and discard the supernatant. Wash the cell twice with PBS.
i. Analyze the stained cells with flow cytometry after
resuspension.
Note:1) the cells can be kept at 4 degree centigrade in the dark, if FCM analysis will not be carried out immediately.
2)
PBS-FCS: PBS with 5% (v/v) fetal calf serum and 0.1% NaN3.
3) You can skip step e & f, if you use fluorescence conjugated primary
antibody.
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