In order to properly run and analyze a flow experiment, a number of different controls are required.
The Rules for Compensation:
- Controls need to be as bright as any sample compensation will be applied to.
- Background fluorescence should be the same for the positive and negative control populations.
- Your compensation fluorochrome must be matched to your experimental color. This means you do not use Alexa 488 to compensate for a GFP sample.
Compensation controls can be set up by either using single-stained cells or beads. To run compensation, you will need an unstained control and a single stained control for each color used.
Example of controls:
For mouse splenocytes stained with FITC, PE, PerCP, and APC conjugated antibodies:
- tube 1) unstained splenocytes
- tube 2) FITC stained splenocytes
- tube 3) PE stained splenocytes
- tube 4) PerCP stained splenocytes
- tube 5) APC stained splenocytes
or if using beads
- tube 1) unstained beads
- tube 2) anti-* FITC stained beads
- tube 3) anti-* PE stained beads
- tube 4) anti-* PerCP stained beads
- tube 5) anti-* APC stained beads
Why do I need these controls?
Spectral overlap between fluorochromes in multi-color experiments needs the use of fluorescence compensation controls. The unstained cells establish the background fluorescence of the experimental samples and are used to set the PMT voltages.
When you record the compensation controls it is important that you have already established the voltages as all the voltages must be the same between all the controls. Each compensation tube must have a population of brightly stained cells or beads to accurately determine the spill-over values. Never use dim stains- if your antibody staining is dim you can use another antibody with the same fluorochrome to set up the compensation.
Several vendors sell beads specifically for use as compensation controls. Some Compensation Beads that are useful:
- Flow Cytometer Calibration Beads for AcGFP/EGFP and mCherry : beads for compensation of GFP and mCherry
- ArC™ Amine Reactive Compensation Bead Kit : beads for compensation of LIVE/DEAD® Fixable dead cell stain kits.
- Collect enough events (beads or cells). The default is 10,000 which is good for beads, but for cells its a good idea to increase this to 50,000
- Sample on LOW (remember that running on medium or high increases co-incident rates and the % CV of your peak. This results in a broader peak which can be harder to set the region on)
- Once the voltages are set for compensation never change them for that experiment
- Tandem dyes need to be compensated for each lot and each day as they break down and vary between lots
- Your fluorochrome mixes will indirectly impact how well you can resolve populations
- Your compensation controls need to be as bright as your brightest cells.
- Do NOT do manual compensation. You can use Diva software during acquisition or afterwards or you can do compensation using the 3rd party software.
- It is recommended to check the compensation calculations by looking at the means of the negative and positive stains before and after compensation
In order to analyze and interpret your data, you will need to know where to set your gates. Gating controls help you establish this.
FMO’s= Fluorescence minus one
FMO is the current standard standard for gating controls. FMOs are used to identify spread based on the colors in a panel. As the name suggests, an FMO contains all fluorochromes within a panel except one. You can read more about this here. The seminal paper by Holden T. Maecker and Joseph Trotteron this can be found here.
So using the sample above with splenocytes stained with FITC, PE, PerCP, and APC conjugated antibodies, your FMO controls would be:
- tube 1) PE, PerCP, APC stained splenocytes
- tube 2) FITC, PerCP, APC stained splenocytes
- tube 3) FITC, PE, APC stained splenocytes
- tube 4) FITC, PE, PerCP stained splenocytes
Tube 1 (FITC FMO= no FITC stain) is used to set the gate for FITC positive cells. Tube 2 (PE FMO) sets the PE gate. And so on, and so forth.
Why do I need these controls?
More and more reviewers are looking for and requiring FMOs for publishing flow data. FMO are a better control than using isotype controls (isotype controls only tell you how well you blocked your cells). They are important to accurately identify between positive and negative populations, especially when you have dimly expressed antigens or a continuous distribution of staining. The example below highlights the difference an FMO can have on your gating strategy: