The following controls are absolutely essential to flow cytometry, and should be prepared for every experiment:

• A tube of cells with no fluorochromes added with the exception of a viability dye (know either as an unstained or untransfected control, depending on your experimental system). The cells should be subjected to all the resuspensions and washes that your stained samples require. This control is used to set the voltage level for the scatter and fluorescence channels.
  
  If this is the first time you are running this assay, don't be stingy with the number of cells you allocate to this control - it can take a reasonable amount of time to adjust the voltages correctly, and this must be done in real time (i.e. with cells running through the machine).
   
• Positive control tubes, each stained with a single colour for every fluorochrome used. These act as your compensation controls and are used to measure the spillover between fluorescence detection channels (which produces false positive signal). Spillover is dependent on the setup of the machine for that day, not the experiment, so you cannot simply use the compensation settings from your last experiment - it must be calculated each and every time.
  
  Your sample MUST be positive for the markers used in your compensation controls, even if you are not intending to sort or analyse your sample based on those marker. For example, if you are planning to detect a dim and rare population of CD34+ stem cells in bone marrow, it is more useful to stain for CD45 in your compensation control - as long as the fluorochrome is the same, your compensation will be valid (with the exception of tandem dyes, which exhibit batch-to-batch variation).
  
  If your compensation control tubes do not contain positive staining that is as bright or brighter than the staining in your test samples, we cannot set up the cytometer properly and will not be able to determine the boundary between positive and negative staining with certainty.
  
  The only time you do not need to prepare compensation controls is if your assay has one colour only (plus or minus a viability dye - spillover does not need to be calculated for viability dyes).

The following are additional useful controls to test for false-positive staining:

• The ultimate control is to use a variant of your cells of interest which does not express the antigen you are staining for. This is rarely practical, however, so you will more likely want to use either secondary only or isotype controls (see below).
   
• If your antibodies are not directly conjugated, it is a good idea to include controls in which your primary antibodies have been omitted (often referred to as "secondary only" controls). This control will eliminate the possibility that any positive staining may be due to your conjugated antibody binding to your cells non-specifically.
   
• Isotype controls can be useful during initial experimental runs as a control against false positive staining by the antibody isotype, rather than antibody-antigen interaction. This is also the only control available when using directly conjugated primary antibodies (unless you have access to the cell line control outlined in point one).
  
  Important note: when staining with an isotype control antibody, you must make sure that you add the same amount of isotype control antibody as you have in your test stain - take careful note of the starting concentrations of your reagents, they often differ!
   
• Fluorescence minus one (FMO) controls. When staining with a large number of colours (i.e. more than three) you may see cells that look like they have stained positive but are in fact artefacts due to spillover between fluorescence channels, revealed by applying compensation to the data. For this reason it is useful to run controls which are stained for all of the colours in your assay except one - this will allow you to set the boundary between negative and positive staining with certainty.

Controls for flow cytometry experiments are as important as they can be confusing, so do not hesitate to ask a FlowCore staff member if you need help designing your assay.