Written by James McCracken, Ph.D.
As cells die, the membrane becomes permeable.
This allows for antibodies to penetrate the cells, which can now mimic live cells. For this and other reasons, it’s important to remove dead cells from further analysis during your flow cytometry experiments.
For example, let’s say you merely need to generate an accurate cell count. If you fail to remove your dead cells first, you might think you’re seeding 10,000 cells, but in reality only 7,000 of your cells are actually viable.
Since the dead cells in your sample will not divide, your culture will take extra time to reach the needed level of confluence, ruining your experimental timeline and weekend plans. Or maybe you’re sorting cells for a downstream functional assay, stimulating sorted T cells with antigen and measuring production of IFN. If you failed to remove your dead cells first, you could end up with different percentages of dead cells in one sorted sample versus another sample. This means you will end up with fewer vital cells in one set of wells versus another, altering your results and negatively influencing your interpretation of the data.
In one final example, let’s say you’re simply staining surface antigens in a population of cells. The problem here is that dead cells take up antibody very readily. If you failed to remove your dead cells beforehand, gating on your population of interest will be exceptionally difficult. To make matters worse, both dead cells and apoptotic cells are highly autofluorescent. These last two issues, in particular, will become even more troublesome if you are using dim markers or rare cell types.
Why You Need To Remove Dead Cells
Dead cells should be removed from all flow cytometry experiments that aim to evaluate live cell lineage and functionality.
Below are two simple examples of why you need to remove non-viable cells prior to implementing your flow cytometry gating strategies.
The two plots display mesenchymal stromal cells (MSC) produced in a Good Manufacturing Practices lab. The lab in question is very good at producing these cells from bone marrow and routinely generates 100% CD45-negative cells after their analysis.
In the panel on the left, the sample is stained with a dead cell marker only. Here, you can easily see what appears to be CD45 contamination of their MSC product in the dead cell (dead cell marker-positive) fraction. This would NOT be possible without the dead cell marker. Also, as discussed above, the left panel reveals the higher levels of autofluorescence in the dead cells. Finally, by adding anti-CD45 antibody to the sample (right panel)you see both the autofluorescent population, as well as a separate CD45-positive population that’s taking up the antibody.
Fortunately for scientists and flow cytometrists like you, there are multiple ways to label and identify dead cells so they can be removed from your flow cytometry analysis and cell sorting experiments.
3 Dead Cell Reagents To Improve Your Data Analysis
There are several methods for analyzing live, dead, and apoptotic cells by flow cytometry. These methods can be divided into three reagent classes, including classic DNA dyes, amine reactive dyes, and vital dyes.
With the following three options of live dead cell reagents available to every scientist and flow cytometrist, you should have little trouble finding a dye that fits into your antibody panel and your flow cytometry assay conditions overall. Adding the right reagent will result in increased quality in your data and increased confidence of your conclusions by both you and those reviewing your grants and papers.
1. Classic DNA dyes.
Classic DNA dyes are exactly that—classic. They are the first type of live dead cell dyes that most scientists and flow cytometrists consider for their experiments. Examples of these dyes include the Sytox dyes, DRAQ7, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD). PI and 7-AAD in particular have a long history in flow cytometry applications. This is because the method of action of PI and 7-AAD are very similar. They are both DNA binding dyes that are membrane impermeant, meaning living cells with intact membranes will exclude these dyes and exhibit little to no fluorescence. Here are the pros and cons overall…
Pros: Classic DNA dyes are easy to use and typically added at the end of staining, which means they require minimal incubation. These DNA binding dyes are also inexpensive, meaning you should have little trouble convincing your boss to buy them.
Cons: Cell impermeant dyes are not appropriate for fix perm staining applications. You will also be limited in excitation and emission selections compared to other dead cell reagent options. You must also use a dead cell compensation control for experiments using these dyes. A good way to prepare such a control is by heat killing a sample of your cells at 70°C for 30 minutes prior to adding the dye.
2. Amine dyes.
Amine dyes are one of the greatest flow cytometry inventions since automatic compensation. First, they are fixable, so whether you’re traditionally staining your cells or fixing and permeabilizing your cells, the fluorescence is maintained. As a result, you can batch a large number of samples while still keeping your flow cytometry best practices intact.
Second, amine dyes are available in a wide range of excitation and emission profiles, making them extremely easy to work into today’s increasingly multi-parametric and multicolor assays. Amine dyes are also membrane impermeant, but rather than binding DNA, they work by binding the amine groups of cellular proteins. Live cells with intact membranes will allow the dye access only to the few amines on the cell surface, while dead cells will allow the dye access to the many more amines on proteins inside the cell, resulting in higher fluorescence.
Pros: There is a wide selection of amine dyes from multiple manufacturers so you can fit these into any flow cytometry antibody panel with ease. Amine dyes are also fixable so they can be easily integrated into batching protocols. Finally, amine-reactive beads are now readily available for use as your dead cell marker compensation control.
Cons: While amine dyes are ideal for your fixation and permeabilization experiments, their use will add time to your fixation protocol. In addition, amine dyes are more expensive than the other reagents listed here. Titration of your amine dyes can reduce costs, but titration must be done carefully to minimize the number freeze-thaw cycles you subject the reagents to. Most importantly, you must remember to label your cells with amine reactive dyes only in the absence of free protein, otherwise you’ll stain the protein in your solution, not the cells of interest.
3. Vital dyes.
Instead of binding to DNA, like the classic DNA dyes, or to protein like the amine reactive dyes, this third class of reagents measures viability by fluorescing when acted upon in metabolically active cells. Calcein acetomethoxyis membrane permeable, yet, due to its attached acetomethoxy group , does not fluoresce. However, once inside a metabolically active cell, cellular esterases cleave the acetomethoxy group yielding calcein. Once free, calcein readily binds intracellular calcium and fluoresces brightly green. As a result, viable cells appear bright green, while dead cells do not.
Pros: Like classic DNA dyes, calcein is easy to use and fast acting. All you have to do is add the reagent to your sample, incubate for a few minutes, and then analyze. Also like classic DNA dyes, this vital dye is inexpensive. Just remember to titrate the reagent before adding it to your cells of interest, as each cell type will have a different optimal staining concentration.
Cons: Since calcein requires cleavage by active cellular esterases, you cannot easily use this dye in your fixation and permeabilization experiments. You are also limited in terms of excitation and emission choices. As a result, working this vital dye into your multicolor experimental and control panels can be difficult.
The addition of a viability dye is essential for good polychromatic flow cytometry. With the above selection of dead cell reagents, you should have no difficulty fitting this marker into your flow cytometry antibody panel and instrument. Here’s the overall lesson—NO cell preparation is 100% viable. Therefore, the consequences of dead cells masquerading as live cells can result in an overestimation of rare events or the identification of cells that don’t really exist in nature. The only way to prevent this is to remove these dead cells from your final analysis using one of the above reagents.
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Latest posts by James McCracken, Ph.D. (see all)
- What Is The Coulter Principle And Why You Need To Obtain Complete Blood Counts By Flow Cytometry - December 9, 2015
- 3 Reagents For Identifying Live, Dead, And Apoptotic Cells By Flow Cytometry - November 11, 2015
- Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments - October 14, 2015