By Ryan Hamnett, PhD
Flow cytometry uses light to characterize and measure heterogenous suspensions of cells based on their physical characteristics and fluorescence. Flow cytometry typically uses antibodies conjugated to fluorophores to target extracellular markers in order to define cell populations. Appropriate controls are essential to ensuring the accuracy and reliability of flow cytometry experiments, and can assist with troubleshooting in case of spurious results.
| Control | Type of control | Description | Purpose | Notes |
|---|---|---|---|---|
| Unstained control | Negative | Run particles with no antibody or fluorescent marker | Provides background level of signal to set voltages and gates appropriately | |
| Isotype control | Negative | Stain sample using antibodies of the same host, Ig subclass and conjugate, but raised against an antigen not found in the sample | Identifies non-specific binding such as due to Fc receptor binding | |
| Isoclonic control | Negative | Stain sample with both the specific conjugated antibody and an excess of specific non-conjugated antibody | The excess non-conjugated antibody will occupy the majority of the specific binding sites, so the conjugated antibody can only bind non-specifically | A lack of fluorescent signal will indicate that the conjugated antibody is not binding non-specifically |
| Single color compensation control | Negative | Stain sample with only a single fluorescent marker | Identifies the extent of spectral overlap by revealing how much signal from one marker is detected by each detector | Each control should contain both a positive and a negative cell population. Control samples should match the experimental samples as closely as possible: same cell type, expressing the same target antigens at a similar level, same fluorophore. Compensation beads can be substituted if necessary, but they must be as bright as the experimental sample. |
| Fluorescence minus one (FMO) control | Negative | The full experimental panel of antibodies apart from one is used to stain samples | Establishes how fluorophores in a panel spill over into one channel. Allows gates to be set appropriately by identifying where a negative population spreads to | FMO controls should be included for each fluorophore in the panel when starting a new multiplexed experiment. With more experience, not all parameters will require a FMO control |
| Cell viability control | Negative | Include a stain in panel that distinguishes live cells from dead cells | Allows exclusion of dead cells from analysis, which can have high autofluorescence and non-specific binding | |
| Secondary antibody control | Negative | Stain cells only with a secondary antibody while omitting the primary antibody | Establishes extent of non-specific binding due to the secondary antibody | Not necessary if using only directly conjugated primary antibodies |
| Negative biological control | Negative | Stain a sample known not to express the antigen of interest | Helps to identify background signal from negative populations | Samples can include cell types that do not naturally express the antigen, or cells which have had the antigen knocked out or knocked down |
| Positive biological control | Positive | Stain a sample known to express the antigen of interest at high levels | Establishes positive signal profile to compare with experimental samples | Samples can include cell types that naturally express the antigen, or cells which have been transfected to overexpress the antigen |
Table 1:Key controls to run alongside a flow cytometry experiment.