Experimental Controls Optimization

Selecting the Right FACS Controls

One of the first things to consider when designing your flow cytometry experiment is which controls to use. There are many sources for error and variability that can be introduced into experimental measurements, and it is important to control those points. The purposes of these controls are to ensure the flow cytometry instrument is functioning properly, that compensation is correctly set, that appropriate gates can be drawn, and the correct interpretation of the data can be obtained.

Use this table to help decide which controls are appropriate for your flow cytometry experiment:

Control What to include Purpose Notes
Unstained control Unstained cells (incubated in parallel with your stained samples) that are fully processed without addition of any antibodies. To control for background derived from auto-fluorescence, and to set the voltages and negative gates appropriately. Used as an additional negative control. Comparison to beads can help to determine the relative amount of autofluorescence. Try using a different excitation source if autofluorescence levels are high.
Isotype control Cells incubated with isotype control antibodies (antibodies usually raised against an antigen that should not be present in your cells). To determine nonspecific binding of the primary antibody. The isotype control should match:
-The host species
-Ig subclass (IgA, IgG, IgD, IgE, or IgM) of the primary or secondary antibody
-Conjugated to same fluorophore as the primary antibody
Internal negative control Population of cells that do not express the antigen of interest and are fully processed. To avoid false positives resulting from nonspecific antibody binding. Negative control cells are not always available. Ideally, the fluorescence intensity of the internal control should be the same as the unstained control.
Positive control Cells known to express your target of interest. To avoid false negatives resulting from a bad antibody. Positive control cells are not always available.
Compensation controls Compensation controls must match the exact experimental fluorochrome (with a similar brightness). The controls need to be at least as bright or brighter than any sample the compensation will be applied to, and background fluorescence should be the same for the positive and negative controls. Allows you to remove (compensate for) spectral overlap. Spectral overlap should be compensated for every fluorophore used. Single staining in multicolor flow cytometry is important due to spectral overlap between different fluorophores. Be sure to collect enough events to be statistically significant.
Viability control Common viability dyes to identify dead cells include DNA dyes or protein binding dyes. Exclusion of dead cells using viability staining means less non-specific binding and easier identification of positively stained populations. Using a live/dead stain to remove dead cells can improve your staining. Dead cells have greater autofluorescence and increased non-specific antibody binding, leading to false positives and reducing the dynamic range.
Fc blocking control Fc blocking reagents added to staining procedure; a commercial Fc block can be used. To ensure that only antigen specific binding is observed (Antibody binding via Fc receptors can lead to false positives and data that cannot be interpreted). Alternatively, the serum of the host primary antibody can also be used. For example, if your antibody is of a mouse isotype, you could use mouse serum to block non-specific binding of immunoglobulins/antibodies to the Fc receptors. For purified PBMCs, 10% human serum can be used.
Fluorescence minus one (FMO) controls The experimental cells stained with all the fluorophores minus one for each fluorophore in your multicolor panel. To detect influence of fluorescence spread within detection channels (especially with brighter fluorophores). Important for building multicolor flow cytometry panels and helps determine where gates should be set.
Secondary antibody control Cells incubated only with the secondary antibody. To determine nonspecific binding of the secondary antibody. Only necessary if using a secondary antibody.

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