There are a variety of fluorochromes broadly used in flow cytometry. It is critical to choose the right ones for your flow cytometry assays. To facilitate your fluorochrome selection, we here outline a few key considerations for your reference.
1. Antigen abundancy
(1) Highly expressed antigens can choose almost any fluorochrome
(2) Low-level expressed antigens require fluorochromes that offer a high signal-to-noise (S/N) ratio, such as PE and APC.
2. Autofluorescence
(1) Each cell population exhibits varying levels of autofluorescence.
(2) Autofluorescence can be seen across all fluorescence channels, but its intensity rapidly diminishes at longer wavelengths.
(3) For cells with strong autofluorescence, opting for fluorochrome with longer emission wavelengths (e.g., APC) can yield a better S/N ratio;
(4) For cells with weak autofluorescence, fluorochromes with longer emission wavelengths no longer significantly improve the S/N ratio. FITC will be a better choice.
3. Low FRET efficiency of tandem dyes
A tandem dye is a conjunction of two fluorescent molecules - a donor and an acceptor - that are covalently bonded. The donor fluorophore absorbs light energy of a precise wavelength, then transfers this to the acceptor through a process called Förster resonance energy transfer (FRET), or fluorescence resonance energy transfer. A robust signal will be observed in the acceptor channel when FRET efficiency is high, while a weaker signal will be detected in the donor channel. Given that FRET efficiency is not always 100%, a 'bleed-through' or spillover of the signal in the donor channel often occurs. However, if you notice a strong signal in the donor channel and a weak signal in the acceptor channel, the following factors should be considered, which cause low FRET efficiency.