Flow cytometry is a common technique used for a wide range of applications including characterization, detection of the cells, tracking cell progression through protein expressions, or identifying changes in cell markers. In every flow cytometry assay, the viability staining is performed together with the markers of interest.

Why is live & dead cell identification so important?

Viability staining in flow cytometry assay helps guide you to:

• Separate live and dead cells population in the analysis
• Eliminate autofluorescence signals that originate from dead cells
• Decrease non-specific antibody binding from dead cells
• Avoid misleading interpretation of the results

What are the common viability dyes used in flow cytometry?

• Propidium iodide (PI)

Propidium iodide is a membrane-impermeable, reversible binding dye which can bind to both double-strand DNA and RNA of dead cells or cells with compromised membranes, without base preferences. However, PI has a broad emission spectrum (Ex:488 nm and peak EM: 620 nm) which overlaps with FITC and phycoerythrin (PE) and it is also shown to leach from the cells at a fast rate.

• Aminoactinomycin (7-AAD)

7-AAD is usually used as an alternative to PI dye, it can only bind to GC-rich regions of double-stranded DNA. This dye is also membrane impermeable so the dead cell can be easily discriminated from the live/contact cells. Unlike PI, 7-AAD has been shown to persistently bind to the DNA after the staining process, the signal can stay longer than PI and has less emission spectrum overlapping to FITC and PE, allowing the multi-colour flow cytometry analysis can be easily performed.

How to get better flow cytometry results?

Using the right viability dyes can have an impact not only on your flow cytometry results but your overall contribution to the validity of your hypothesis. With the limitations of PI, using ghost dyes will be a suitable alternative to get clear and reliable flow cytometry analysis.

• Ghost dyes ^TM

This amine-reactive dye can covalently bind to the amines in both intracellular amines and surface membrane amines, allowing dead cells to show greater fluorescence than live cells. Ghost dyes ^TM is an alternative to conventional dyes with better advantages

• Withstand subsequent washing, fixation, and permeabilization process
• The irreversible binding provides a longer stable signal
• Available in wide ranges of excitation and emission spectra, allow easier multi-colour staining
• Ready-to-use format and easy to incorporate into staining protocols
• Comparable to BioLegend’s zombie dyes and eBio’s LIVE/DEAD kits with a more economical option

Selecting the most suitable dyes for viability staining in your flow cytometry assay is an important step prior to moving forward to further analysis. The suitable dyes can provide accurate and clear results which facilitate your interpretation.

Need support to find the right dyes for your study? Contact us.

References

• N. Falzone, C. Huyser, D. R. Franken. 2009. Comparison between propidium iodide and 7-aminoactinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome. https://doi.org/10.1111/j.1439-0272.2009.00949.x
• Bio rad. (2022). Live/dead Exclusion. https://www.bio-rad-antibodies.com/flow-cytometry-live-dead-exclusion.html
• Ghost Dyes™ & Cell Viability Reagents. TONBO^TM A CYTEK® Band. https://tonbobio.com/collections/ghost-dyes-cell-viability-reagents