Live-or-Dye™

The Live-or-Dye™ fixable viability stains, including Live-or-Dye NucFix™ Red, are dead cell stains that are amine-reactive. This property of the dyes makes staining compatible with fixation, but challenging in multi-cell layer systems such as 3D, Matrigel® or organoid cultures. As the dyes are reactive, the majority of the staining would be restricted to the outer/ exposed cell layer/s. Longer incubation times to allow the dye to penetrate deeper in to the cell layers would generally not be helpful as the labeling reaction is short-lived (15-30 min).

See our recommendations for dyes suitable for 3D cultures or organoids.

For fixable nuclear dead cell stains, other options to consider would be NucSpot® Far-Red. Designed to be an improved alternative to the more common dead cell stain 7-AAD, the dye is fixable to a degree. Although generally more suitable for flow cytometry as its spectral properties are not appropriate for standard fluorescence microscopy, imaging using a confocal may be better as the Ex/Em can be more appropriately matched. We would also recommend imaging soon after fixation and not storing the sample for extended periods post-fixation as staining would deteriorate over time.

The viability dye PMA may be another possibility. PMA, a photoreactive version of propidium iodide (PI), is generally used for viability PCR of bacteria. However, it can be used with mammalian cells (based on internal customer feedback) as a fixable dead cell stain for flow cytometry. PMA is not fluorescent until it binds DNA. Subsequent exposure to bright light activates the dye and causes it to photo-crosslink to DNA, making it fixable. Incubation of the dye with cells would need to be done in the dark, and the excess dye washed away before exposing to light for photo-crosslinking. Optimization for light exposure sources (LED lights can be use to avoid heat) and light intensity (bright enough to crosslink the dye without photobleaching; generally close proximity to a bright LED or halogen lamp is required, room light is not sufficient) may be required.

Live-or-Dye NucFix™ Red is a reactive dye and most cell culture media, even if they are serum-free, contain a high concentration of reactive groups that would react with the dye, rendering it unreactive. While the dye would still get inside the cell and staining would be specific to the nucleus (the dye is fluorogenic and should only fluoresce when bound to DNA), it would not remain fixable.

PMA and PMAxx™ are photoreactive, cell impermeant DNA binding dyes that may be a better choice for fixable dyes for cell staining in medium. The dyes need blue light for photoactivation. Standard blue LEDs can be used for photocrosslinking of the dyes to DNA. If long-lasting fixation is not a requirement, and if the cells will not be undergoing extensive permeabilization, more fixable dead cell stains like NucSpot® Far-Red or 7-AAD  may be suitable options. Though the signal to noise is not as good as in unfixed cells, these dyes are more fixable than other dead cell dyes such as propidium iodide (PI).

Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III. Wash the cells several times to remove unbound Ethidium Homodimer III before fixation.