Question 9 of 20 Fundamentals Practical
Why include a viability dye in a flow panel, and how do you choose one?
Short answer
Dead cells bind antibodies nonspecifically and can create false positive events, especially in rare populations. Use a DNA dye such as propidium iodide or 7-AAD for live, unfixed cells, and a fixable amine-reactive dye whenever the cells will be fixed or permeabilized.
Why
A dying cell has a leaky membrane and sticky contents. It picks up many antibody conjugates for reasons unrelated to its antigens. When the population of interest is 0.1 percent of events, a few dead cells can be most of what sits in the gate.
DNA dyes such as propidium iodide and 7-AAD cannot cross an intact membrane, so they stain only dead cells. They must stay in the buffer, and they wash out or redistribute after fixation, as Perfetto and colleagues describe. Amine-reactive dyes bind proteins.
A live cell exposes only its surface amines and stains dimly, while a dead cell lets the dye in and stains brightly. The covalent bond survives fixation and permeabilization, which makes these dyes the default for intracellular staining. They react with any protein, so they are stained in protein-free buffer before serum or albumin is added.
Pick a color that fits the panel's spare detector and does not spill heavily into a key dim marker. For example, a panel for intracellular cytokines in T cells needs a fixable dye, applied before fixation. Viability dyes have limits.
Early apoptotic cells can still exclude both dye classes, so an annexin V or caspase stain is needed when apoptosis itself is the question.
A strong answer covers
- Dead cells bind antibodies nonspecifically
- DNA dyes for unfixed cells, kept in the buffer
- Amine-reactive dyes survive fixation
- Stain amine dyes in protein-free buffer
- Viability dyes do not detect early apoptosis
Common mistakes
- Using propidium iodide in a panel that will be fixed and permeabilized
- Staining with an amine-reactive dye in a buffer full of serum protein
- Relying on scatter alone to remove dead cells before gating a rare population
The interviewer might ask next
How would you build a compensation control for an amine-reactive viability dye?