Like any other living thing, each cell has its own life cycle. When studying or sorting cells with flow cytometry, researchers focus on living cells. Dead cells can cause issues in the flow cytometry process as well as downstream assays. One way to reduce the concentration of dead cells is through live/dead staining with a fixable viability dye.
The main issue with dead cells is the loss in viability. Cell viability refers to the ratio of healthy cells to damaged or dead cells in a sample. Because these dead cells no longer perform the function(s) that they did while alive, they do not contribute to downstream results.
When performing flow cytometry, a method of categorizing cells through a variety of physical characteristics (size, light scatter, density, etc.), dead cells can be misidentified. Dead cells also release cell debris when their membranes rupture. This extracellular debris can clump up to cause inaccuracies and more cell death.
Regardless of how the cells died, it’s important to extract as many as possible. By removing these dead or damaged cells, scientists can ensure that their sample viability is high. A high starting viability optimizes flow cytometry, prevents additional cell death, and reduces inaccuracies caused by cellular debris.
Dead cell exclusion requires precise, gentle removal tactics to avoid doing harm to other healthy cells in the sample. The first step to this removal is to correctly identify which cells are damaged.
Scientists use cell viability fluorescence dyes to identify healthy or unhealthy cells in a sample. These dyes harness fluorescent substances that interact with specific cells causing them to light up, thus revealing their location.
The fixable viability dye process begins by isolating a specific cell sample from a heterogeneous mixture through cell separation. Once the desired cell type has been isolated, researchers must decide which strategy they want to use to measure and remove dead cells. Viability dyes enable this to occur in one of three ways:
The specific dye you use depends on the context of your experiment and the cell populations you are working with.
There are a variety of different viability dyes, or stains, available to researchers. The most commonly used substances bind to different nuclear portions contained in DNA within cells. Whether or not a dye can pass through a healthy cell membrane heavily contributes to the cells that it is capable of illuminating.
DAPI, or 4’,6-diamidino-2-phenylindole, is a popular fluorescent blue dye that can be used to identify cells. It binds to DNA regions that are rich in adenine-thymine nuclear combinations. DAPI is considered to be a cell impermeant, meaning it typically cannot pass through the membrane of live cells. However, in high concentrations, DAPI will also mark live cells. DAPI live-cell staining will also label dead cells unless used with a counterstain. Depending on the quantity this dye can be used to identify dead cells or all cell types in a sample for counting.
DAPI is useful when used with a counterstain because it can sometimes leak into healthy cells. It is also beneficial to wash a sample before using DAPI. Taking the necessary steps before staining will help to minimize possible staining errors.
Hoechst stain is yet another fluorescent blue dye that can be used for live-cell staining. It is more permanent than DAPI and takes a lower concentration to pass through a healthy cell membrane. The Hoechst staining protocol requires only one-tenth the quantity of DAPI to stain live cells. Unfortunately, while Hoechst can be used for nuclear staining in microscopy, it should not be used for flow cytometry. The stain can interfere with lasers in the flow cytometer and cause more than one to be excited.
One of the simplest and most effective methods of measuring viability in a sample is the staining assay trypan blue. Trypan blue is a fluorescent dye that targets and identifies dead cells. This dye cannot pass through healthy membranes and cannot bind to their DNA. This stain is commonly used with flow cytometry to eliminate false positives during cell counting.
After a cell dies of either necrosis or apoptosis, trypan blue will penetrate the cell and illuminate it with fluorescent blue color. The mechanism relies on the dye not interacting with cells unless the membrane is damaged and a negative charge that allows it to bind to DNA.
Another popular impermeant fluorescent exclusion dye used with flow cytometry is propidium iodide (PI). PI is a fluorochrome that binds to double-stranded DNA by intercalation between base pairs. Typically used to evaluate levels of apoptosis in a variety of experimental models, PI assays can improve viability for flow cytometry by allowing researchers to filter out dead cells and cell debris.
When cells die, their DNA fractures and disperse throughout the sample. If allowed to pass through the flow cytometer these cell fragments may still activate fluorescent lasers, causing false positives to be recorded. PI stain can precisely identify these damaged cells for removal in around two hours, a small price to pay for more accurate results.
A multitude of fluorescent dye cytometric assays can be harnessed to improve sample viability for flow cytometry. Beyond just identifying and labeling dead cells, additional steps must also be taken to remove cell debris or prevent cell death to minimize inaccuracies in an experiment.
One of the simplest ways to prevent cell death and therefore increase sample viability is by following cell separation protocols. Before cells can be measured by a flow cytometer, they are typically isolated and purified from a heterogeneous mixture of some sort. For example, T cells and B cells are often isolated from whole blood samples. Cell separation helps to extract T cells from other residual blood cells, so the flow cytometer does not have to deal with as many undesired contaminants.
Certain cell separation techniques can contribute to cell death in a sample because they rely on harsh physical or magnetic forces. Methods like centrifugation or magnetic cell sorting can be detrimental to the throughput of fragile cell populations. While the cells that die during separation can still be removed with the proper viability dye and exclusion methods, the process will take longer and prove more difficult with a larger quantity of dead cells. Not to mention the fact that research experiments benefit from having as many healthy cells as possible. To optimize a flow cytometry analysis assay, we recommend you use a gentle cell separation method such as Akadeum’s buoyancy activated cell sorting (BACS).
Akadeum has developed an innovative method of cell separation that relies on the natural properties of tiny, buoyant bubbles to quickly and gently isolate target cells. Some cell separation methods require you to purchase additional pieces of expensive equipment to perform an experiment, BACS only requires a small microbubble kit that can be stirred into any container.
The main benefit of using BACS with flow cytometry is the reduction in cell death. The soft, but strong nature of microbubbles removes additional contaminants by floating them to the top, leaving the enriched substance untouched at the bottom of the solution. For more information on the interaction with flow cytometry, read our page on Using Microbubbles to Improve Flow Cytometry.
Akadeum’s microbubbles can be used for fast, simple, and gentle cell isolation to prepare a sample for cell separation. Akadeum is also developing a Dead Cell Removal Kit that can be used to remove cells stained with specific markers. This product can be used to supplement other separation methods and increase sample viability for flow cytometry or other downstream assays. If you’re interested in our products, a commercial partnership, or are simply curious about microbubble technology, contact Akadeum Life Sciences today.