Hello,
I'm trying to select fluorophores for a large panel of flow cytometry, so I'm wondering what is the maximum degree of overlap between two fluorophores that is considered acceptable? Thanks.
Hello Vincent
First and foremost, selection of fluorophores will depend on the number and types of lasers and filters present in the flow cytometer. When you are using a panel of fluorophores it is always better to distribute them as widely as possible across the lasers and filters to reduce the interference between them. It will be better to use bright fluorophores like PE or APC for low expressed proteins and for the highly expressed proteins one can use dim fluorophores like Alexa 700 or Per CP.
If you wish to increase your panel size you can make use of tandem dyes wherein two fluorophores are conjugated to the same antibody. The transfer of energy between the two fluorophores takes place by FRET. The second fluorophore in the tandem dye emits light at a higher wavelength than the first fluorophore. So one can have more readouts using the same laser. APC and PE are common donor fluorophores for tandem dyes such as APC-Cy-7and PE-Cy 5.5 respectively. To get consistent results with tandem dyes use conjugated antibody from the same batch.
You can avoid the need for compensation by using fluorophores with distinct non-overlapping spectra. But this is too difficult when the panel size increases. Therefore compensation controls are essential to make sure that detected light is from the right fluorophore.
Regards.
Dear Vincent !
You look also at this publication:
https://www.thermofisher.com/ru/ru/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-71/bioprobes-71-flow-cytometry-panel-design.html
The SI can be useful for comparing histograms of cell populations stained with different fluorescent conjugates of the same antibody (Table 1, Figure 3).
Figure 2. Comparison of Stain Index (SI) and signal-to-noise ratio (S/N). An illustration of two fluorophores with the same S/N but different SI due to different widths of the negative peak (narrow W1 vs. wide W2). Because the width of the negative peak affects the separation of the positive and negative signals, SI is the preferred statistic when comparing fluorophore brightness.
Table 1. Staining Index for different fluorophore conjugates
This is something that is likely dependent on many factors, such as signal to noise ratio, available lasers, filters and their bandwidth and the fluorophore available for your targets of interest. AS Malcolm detailed, Fluorophores with the brightest stain index (such as PE) are best used for cells with the lowest antigen expression or the smallest subset, whereas dimmer fluorophores are more suitable for more highly expressed antigens. However, when using several colors, compromises may have to be made due to availability of fluorophores, antibodies and flow cytometer configuration. As you increase the number of colors in your panel, you will have to use tandem dyes such as PE-Cy5, PerCP-Cy5.5, APC-Cy7, etc., which increase the emission range from a single laser. This works on the formula
PE-Cy5 + PE overlap) - (PE overlap) = accurate PE-Cy5 results
One has to keep in mind that the brightness of tandem dyes may be reduced by fixation and permeabilization. Moreover, they may also be prone to degradation by light and cellular biological activity resulting in uncoupling and fluorescence emission at two wavelengths, leading to false positives. So ideally, compensation values should be obtained from samples treated in an identical manner to account for this.
Regarding, what degree of spectral overlap is considered acceptable, there is unfortunately no simple answer to this question, since this varies a lot between fluorochromes. You'd need to use compensation controls if you have overlapping spectral fluorophores. Ultimately, you would end up in a situation where if you can subtract the fluorescence of one from another with a suitable compensation % (some combinations require 5% compensations, some other require 30% or more), and get good distinction, that is "acceptable".
Hopefully, this handbook may help provide you with more information about the compensation process: https://info.bio-techne.com/flow-cytometry-handbook.html
I believe some of the links printed below for you may help you in many ways to create a good antibody panel for multicolor flowcytometry
https://www.novusbio.com/novusknowsflow.html
https://www.novusbio.com/spectraviewer
https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20859
BW
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