Processing and staining cells for flow cytometry or cell sorting is a multi-stepped process that can stress cells and cause cell death. Dead or apoptotic cells are troublemakers in cytometry because they can bind antibodies, effectively changing the staining antibody concentration even after titration, which results in suboptimal staining of live cells, and they can autofluoresce, particularly in the green channels, providing false positive signals. Dead cells also do not divide, cannot produce cytokines upon in vitro stimulation, and are often sticky due to extruded nucleic acids resulting is both poor yield and poor purity when sorted. So if you have a large fraction of dead cells in your samples, your results may be significantly skewed or unusable.
Before you even consider how to design your staining panel, you must decide how you will discriminate between live and dead cells. While it is common practice to use light scatter (low forward and side scatter) as an indicator, this method is highly inaccurate so the best approach is to use a viability dye.
What Is A Cell Viability Dye?
A cell viability dye stains either live or dead cells depending on its mechanism of action. Some viability dyes can pass through the damaged cell membranes of dead or apoptotic cells to stain DNA or proteins inside. Other dyes enter live cells and are metabolized, which causes fluorescence. These different types of dyes work under unique conditions, so spend time researching the different dyes to select one that will work with your staining or sorting protocol.
How Do I Know A Dye Will Work On My Flow Cytometer?
Viability dyes function similarly to fluorescent antibodies as they each have a specific excitation and emission wavelength. A wide variety of dyes exist, but be sure to check the specs of your flow cytometer to make sure it houses a laser and detector that are compatible with your viability dye.
How Does A Viability Dye Affect My Staining Panel?
Since viability dyes work at specific excitation and emission wavelengths, including such a dye may prevent you from using a specific fluorophore that has an overlapping excitation/emission spectrum. Take time to consider which viability dye may work best with your staining panel, or do a small pilot experiment with different viability dyes or different fluorophores so you can make the most of your panel.
When Are Viability Dyes Used In My Experiment?
Viability dyes are added at different steps during a cell staining protocol depending on the dye. Some dyes are “fixable” and are appropriate for intracellular staining or protocols where samples must be preserved through fixation. In these cases, the dye is added prior to the fixation and/or permeabilization step and the initial staining pattern is maintained. Other dyes are non-fixable and are appropriate for surface staining, cell sorting, or protocols where a fixation method is not required.