Many dyes can assess cell viability when using. Classic dyes such as such as (PI) and (7AAD) are
- Cell membrane-permeable, intercalating with double-stranded nucleic acids, DNA in particular, of cells whose membranes have become more permeable, a sign that such cells are in cell membrane damaging types of distress, dying or dead.
- Added at the end of the staining protocol, thus requiring little by way of extra time or steps.
An inexpensive, positively charged dye that cannot cross an intact plasma membrane, PI has fairly discreet excitation and emission spectra, excites at 488nm (maximum 535nm) and emits within the 570 to 630nm range (red fluorescence emission).
Full disclosure: For reasons explained below, I preferred 7AAD to PI for flow cytometry cell viability assessment and switched away from both to amine dyes as soon as they arrived on the scene.
- PI’s emission spectrum overlaps a little with that of FITC ( ) and a lot with that of PE ( ). Compensating for PI’s spectral overlap with FITC and PE is much more challenging (1) compared to 7AAD. Having been successfully conjugated to thousands upon thousands of antibodies, these are two of the most versatile, proven workhorses in flow cytometry. PI’s overlap with FITC and PE thus severely limits the scope of a multi-staining flow cytometry antibody panel.
- Need to use dead cell compensation control for PI and 7AAD, usually by heat killing 70oC for 30 minutes an aliquot of the cells being stained, which adds an additional variable to the experiment.
- PI could be genotoxic/mutagenic to cells ( ).
- PI can intercalate with RNA as well ( ), a quality long vastly under-appreciated by regular flow cytometry, since morphological assessment isn’t its strength. OTOH, the newer Imaging Flow Cytometry, which combines flow cytometry with fluorescence microscopy, shows increased cytoplasmic PI staining can lead to higher false positives when using standard flow cytometry protocols (4, 5).
- RNA binding of PI is the reason RNase is used with PI in microscopy ( ). However, treating samples with RNase prior to running them on flow cytometry isn’t a widespread practice (5) since RNase doesn’t penetrate live cells. It also requires fixation, which does not work for PI.
- This brings us to cell fixation and the fact that PI only works when using live, not fixed, cells, a major drawback since running fixed cells is a major advantage of high throughput flow cytometry. Cells are typically fixed after surface staining in order to permeabilize their membranes to additionally stain intracellular proteins. Since PI binds DNA non-covalently, when cells are fixed after staining, dye bound to dead cells’ DNA could dissociate and even stain live cell DNA. After all, fixation destroys cell membrane integrity.
Being able to use them on both live and fixed cells is a major reason for the shift away over the past 10 years from PI and 7AAD towards amine dyes. Amine dyes such as Molecular Probes (Invitrogen-Thermo Fisher Scientific) Live/Dead® dye combinations
- Are also cell membrane permeable, meaning they work on the same principle, only entering cells with compromised plasma membranes.
- However, rather than binding DNA, they covalently bind amine groups of cellular proteins. Such dyes would thus only bind the few amines present on the cell surface of live cells but many, many more on intracellular proteins within cells with compromised cell membranes, resulting in a marked increase in fluorescence in distressed/dying/dead cells.
- Covalent binding renders amine dyes impervious to cell fixation, meaning they remain bound only to amines of intracellular proteins within cells that were already dead to start with and won’t leak out to enter previously live, now fixed cells. This is how they can be used to assess viability even on fixed cells.
- Are available in a wide range of excitation and emission profiles.
- Many vendors offer amine-reactive beads to use as dead cell marker compensation control.
- While greatly outweighed by their benefits, disadvantages of amine-reactive dyes are the extra time and step when staining for fixed cells:
- need to stain with them first before permeabilizing and fixing cells for main staining protocol.
- amine-dye staining step needs to be preceded by a saline wash-out step to remove free proteins to minimize non-specific staining of the solution used to suspend the cells, which would waste the reagent and make far less of it available for binding amines of intracellular proteins within cell membrane-compromised cells.
- Since amine-reactive dyes are washed out before the staining steps are done, they track cells that died during the experiment but not the ones that die during the sorting process, when using flow cytometry to sort out subsets of live cells. For this reason and due to the extra steps, classic DNA-binding dyes such as PI and 7AAD are still preferred when using flow cytometry sorting.
1. Telford, William, Karen Tamul, and Jolene Bradford. “Measurement and Characterization of Apoptosis by Flow Cytometry.” Current Protocols in Cytometry (2016): 9-49.
3. Deitch, ARLINE D., H. O. R. A. T. I. O. Law, and R. deVere White. “A stable propidium iodide staining procedure for flow cytometry.” Journal of Histochemistry & Cytochemistry 30.9 (1982): 967-972.
4. Rieger, Aja M., et al. “Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.” Journal of immunological methods 358.1 (2010): 81-92.
5. Rieger, Aja M., and Daniel R. Barreda. “Accurate assessment of cell death by imaging flow cytometry.” Imaging Flow Cytometry: Methods and Protocols (2016): 209-220.
6. Fried, Jerrold, Amaury G. Perez, and Bayard D. Clarkson. “Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.” The Journal of cell biology 71.1 (1976): 172-181.