For simultaneous labeling, choose the fluorochromes carefully. You want the farthest red-emitting antibody to label the antigen with the greatest density (most receptors per cell).
In flow cytometry, fluorescence is relative. We need a negative control to determine where "positivity" begins.
You will need a tube for each antibody plus the negative control. If you're doing simultaneous labeling, use one tube for each combination of antibodies or controls, but we will probably need single-antibody labeled cells for each combination as well. Confused? Let's talk!
This force and time works well for lymphoid cells. You may have to adjust as required if your cells are different.