For each cell type or condition, you will need the following:

1. Autofluorescence control, i.e. cells only; no antibody.
2. Fluorescence control/conjugate control: cells + conjugated antibody against an irrelevant antigen. (Control and test Abs should be the same isotype.)
3. Samples.

Procedure:

1. Resuspend cells to 10 x 10⁶/ml in PBSAz (PBS + 1% serum or protein + 0.1% NaN3). Distribute 50µL to each tube.
2. 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.
3. Add 10µL conjugated antibody and incubate for 15' on ice. Keep the samples dark. The ideal concentration of antibody should be determined by titration.
4. Top with PBSAz and pellet the cells by centrifugation. (we spin for 5' @ 1800rpm @ 4 C.)
5. Decant the supernatant by inverting the tubes and blotting them (touching the rims to a paper towel several times). Do not disturb the pellet!
6. 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.
7. If the samples must be fixed, resuspend them in 0.5ml of 1% paraformaldehyde and store @ 4 C, dark.

Additional Notes:

• Samples must be delivered in 12x75 mm Falcon 2054 tubes.
• 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.