For each cell type or condition, you will need the following:
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Autofluorescence control, i.e. cells only; no antibody.
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Fluorescence control/conjugate control: cells + conjugated antibody against
an irrelevant antigen. (Control and test Abs should be the same isotype.)
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Samples.
Procedure:
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Resuspend cells to 10 x 106/ml in PBSAz (PBS + 1% serum or protein
+ 0.1% NaN3). Distribute 50µL to each tube.
-
Add 10µL unconjugated homologous Ig to each tube and incubate on
ice for 10'. Use the homologous Ig at a concentration 10 times the test
antibody concentration.
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Add 10µL conjugated antibody and incubate for 15' on ice. Keep the
samples dark. The ideal concentration of antibody should be determined
by titration.
-
Top with PBSAz and pellet the cells by centrifugation. (we spin for 5'
@ 1800rpm @ 4 C.)
-
Decant the supernatant by inverting the tubes and blotting them (touching
the rims to a paper towel several times). Do not disturb the pellet!
-
If the samples are to be read the same day they're stained, resuspend them
in 0.5ml of PBSAz. Keep the samples cold and dark.
-
If the samples must be fixed, resuspend them in 0.5ml of1%
paraformaldehyde and store @ 4 C, dark.
Additional Notes:
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Samples must be delivered in 12x75 mm Falcon 2054 tubes.
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Homologous Ig is the same isotype from the same animal that produced the
test antibodies.
-
Never block with homologous Ig if the epitope to be measured is a FcR!
In place of step #4, top the cells with PBSAz and incubate on ice for 10',
in the dark, inverting frequently. Pellet the cells and resuspend in either
1% paraformaldehyde or PBS.
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