What are the dyes used in flow cytometry?

Dyes Used in Flow Cytometry

Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a variety of fluorescent dyes are used to stain cells and their components. These dyes emit light at various wavelengths when excited by lasers. The choice of dye depends on the specific applications, the lasers and filters available in the flow cytometer, and the need to avoid overlap with other dyes where multiple labels are used.

Commonly Used Dyes

• Fluorescein isothiocyanate (FITC): A bright green fluorescent dye excited by a 488 nm laser.
• Phycoerythrin (PE): A protein from cyanobacteria and eukaryotic algae that fluoresces in the yellow to orange range, excited by 488 nm or 532 nm lasers.
• Allophycocyanin (APC): A protein that emits in the red region, excited by a 633 nm or 647 nm laser.
• Peridinin-chlorophyll proteins (PerCP): A complex of proteins from dinoflagellates with an absorption range that contributes to its use in flow cytometry, commonly excited by the 488 nm laser.
• PE-Cy5: A tandem dye where PE is chemically linked to Cy5, allowing for emission in the far-red range when excited by 488 nm or 532 nm lasers.
• PE-Cy7: Similar to PE-Cy5, but emits at a longer wavelength.

Dye Selection Considerations

Dyes are selected based on the specific application. Factors include:
- Fluorochrome brightness: The inherent fluorescence intensity of the dye.
- Excitation and emission spectra: The compatibility of the dye with the laser lines and filters available in the flow cytometer.
- Size and complexity of the panel: In multicolor assays, the panel size and complexity are important to minimize spectral overlap.
- Fluorochrome-to-protein ratio: For conjugated dyes, this ratio affects brightness and aggregation.
- Cell type and antigens: Certain dyes may bind differently depending on cell types and the antigens being targeted.

Multi-Color Flow Cytometry Panels

In multi-color flow cytometry, several fluorescent dyes are used simultaneously to label different targets. This requires careful planning to avoid overlap in emission spectra, which could lead to misinterpretation of data. Compensation is often used to correct for spectral overlap, which is the process of electronically correcting the fluorescence signal of one dye that is detected in the channel of another.

Conclusion

Fluorescent dyes play a crucial role in flow cytometry, allowing for the simultaneous analysis of multiple parameters. The selection of dyes is a critical step in designing a flow cytometry experiment and can significantly affect the quality and interpretability of the results.