Fluorescence Minus One...

Are gating controls useful in large multicolor panels to give accuracy to the gate assignment.  FMOs are particularly useful in 10+ color assays and when the expression of a positive population is poorly defined from the negative population.

FMOs, or Fluorescence Minus One controls, can be extremely helpful, especially when your target antigen is expressed at low levels, or you are concerned with the amount of spectral spillover from other fluors in a large panel. As the name suggests, an FMO consists of the full panel of antibodies while leaving out the target antibody of interest. In some instances, an isotype control can be used in place of the test antibody, which can provide a more complete picture of background signal, from both non-specific binding and fluorescent spillover.

 

 

For additional information on FMOs and compensation, read through this publication by Mario Roederer.

 

 

Compensation controls...

Are a correction factor applied to each channel in use on a flow cytometer to correct for spectral spillover of unintended fluorophores into their neighboring channels.  Compensation controls aid in accurate gating strategies and the avoidance of artefactual events.

Compensation controls are typically necessary for any flow experiment. Fluorophores emit along a spectrum around the peak emission. As a bandpass filter will “catch” wavelengths within its range indiscriminately, compensation should be applied in order to correct for spectral overlap between fluorophores. Compensation controls are single color-stained controls. They can be prepared as stained cells, however, compensation beads are the most robust reagent to use for this purpose.

It should be noted that high compensation values are not inherently indicative of poor reagents. These values can be directly influenced by voltage changes. What’s more important is that the compensation applied is accurate so that the cell populations can be more easily visualized and gated upon.

The four standard practices listed below help to ensure an accurate calculation of compensation:

  1. The reagents applied to the compensation controls, and experimental samples must be from the same vial of reagent and ideally treated the same until acquired.
  2. The autofluorescence of the compensation control must be identical for both the negative and positive events.
  3. Each compensation control's positive population must be at least as bright as any positive signal in the experimental sample.
  4. To ensure precise compensation, collect enough events to estimate median fluorescence in all channels as well as the negative population.

Compensation Beads...

Are stained with single color reagents that present the machine with enough spectral information from the fluorophore in use to generate an accurate compensation matrix.   

Compensation Controls can be generated using single color stained cells or beads. There are limitations to utilizing cells as compensation controls. They must have enough expression of the antigen to generate enough data points for an accurate calculation of compensation for that fluorophore. There also needs to be enough cells in the sample to distribute amongst all of these different kinds of controls and still have enough for the experiment itself.

The alternative is to use compensation beads. Depending on the vendor, the beads will bind a variety of host isotypes and can be quickly mixed with the antibody of choice to create a compensation control. Single color stained cells are ideal for setting the voltages to be applied to the experiment to ensure the positive and negative populations are both within the ideal linear range of detection. Compensation beads are more accurate to populate enough data from the negative and positive populations for the compensation algorithm to calculate. 

 

 

Either compensation beads or single color controls can be suitable for use as compensation controls. The important factors are that there are enough positive and negative events as demonstrated in the histogram using beads. However, since the PMT voltages applied in the assay are determined based on the biological sample to be analyzed, they may not be optimal for very brightly stained compensation beads. In the histogram where beads are employed, the positive peak is still able to be gated on accurately, but is in danger of going beyond the detectable range. In the second example, when using single color stained cells expressing CD44, the cells in this instance will not be suitable since there are not enough negative cells. In addition, the large variance of the MFI+ population can add to additional error in compensation calculation.

Rainbow Particles...

Are useful for ensuring your flow cytometer’s lasers are properly calibrated and aligned. 

First and foremost, it should be noted that Rainbow Particles are not compensation beads, nor can they be used to estimate cell size/number. Rainbow Particles are designed to ensure a flow cytometer is properly calibrated, standardize instruments for longitudinal studies, and evaluate new instrument hardware (e.g., bandpass filters, photomultiplier tubes (PMTs) and lasers). They are excited by any wavelength in the range of 365-650 nm, which includes the violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (633 nm) lasers. They emit in all channels off of those lasers, but at varying intensities.  Rainbow beads can be found in 1 peak, 6 peak and 8 peak options and help characterize the range of sensitivity and linearity of the instrument PMTs and lasers. Rainbow Particles are not compatible with the UV laser.


When you excite fluorophores conjugated to antibodies bound to your cell, you want to ensure that they fall within an ideal range of performance for each laser’s PMT positions. This can be monitored by using the particles and plotting a channel from one laser against a channel from the other laser.

 

 

In the “bad” alignment plot, the violet laser may be underpowered or improperly aligned.

 

As mentioned previously, we offer one, six, and eight peak options, each of which can aid with longitudinal studies to check whether peaks routinely show up at the same fluorescent intensity using a calculation of staining index. For the one peak choices, there are mid-range and  bright intensity peaks. The mid-range option can help represent antigens of moderate expression, while the bright intensity option is ideal for highly expressed targets. Six or eight peak options offer a larger variety of fluorescent intensities. While six peaks is typically enough for most users, the eight peak option can be selected if more sensitivity is desired or the linear range of the PMT or photodiode is expanded.

For more information on the use of calibration beads, take a look at this article by Perfetto, et al.

 

Precision Count Beads™…

Are useful for counting absolute cell numbers in a sample of varying volume.

Precision Count Beads™ are designed for counting the absolute number of cells in a complex population and other particles by flow cytometry. The cell count in the original sample can then be back-calculated. Very few flow cytometers are capable this function, so Precision Count Beads™ are helpful for users without this function built into their cytometer.

Precision Count Beads™ are excited by the violet (405nm), blue (488nm), yellow/green (561nm), and red (633nm) lasers, and emit between 400-800 nm. As the beads have a low forward scatter and high side scatter, this must be taken into account when adjusting the voltage of the scatter channels to ensure the beads are on scale. Vortexing the beads prior to use and using “reverse pipetting” are important for delivering aggregate-free, well distributed accurate amounts of beads.

Precision Count Beads™ can work with all kinds of assays including a lyse, no-wash whole blood assay.

 

 

As shown in the top left plot, Precision Count Beads™ have a very high 
side scatter profile, so the voltage may need to be adjusted accordingly.