Cell Viability Assays

What is cell viability and why is it important?

Cell viability refers to the number of healthy cells within a sample population. It is extremely important in all cell-based assays, particularly those based on Flow Cytometry analysis and sorting, that cell viability is maximised.

Cells should be in an as native condition as possible and so represent the experimental conditions that the researcher is trying to observe. If cells are stressed and cell death occurs, this can lead to erroneous data in the form of shifting of fluorescence signal, nonspecific uptake of staining antibodies and increases in cellular autofluorescence. These changes can make accurate flow cytometry assays difficult, especially if a small number of real positive cells are present or the real shift in antibody positivity is small.

Cell death during sample preparation should always be kept to a minimum.

How do we define cell viability?

Cell “viability” can be defined in many ways:

• Does the cell have an intact cell membrane?
• Is the cell actively respiring?
• Is there a membrane potential, particularly on cytoplasmic mitochondria?
• Is the cell capable of division?

Of these ‘Does the cell have an intact cell membrane?’ is the most fundamental and is what we will focus on in this article.

Once viability has been established, it can be used to:

• Count the number of viable cells
• Exclude dead cells from a sort; it is pointless sorting dead cells to grow on and additionally dead cells may have misleading protein expression in downstream assays.
• Exclude dead cells from a complex immunophenotyping experiment, especially where the numbers of positive cells are very few. Dead cells can give false positive results; dead cells may stain with cell markers non-specifically or have a different autofluorescence spectral signature.

How can we determine if the cell has an intact cell membrane?

To determine if a cell has an intact cell membrane, we typically utilise specific dyes which do not normally pass through healthy cell membranes. If these dyes can permeate the membrane and accumulate in the cell it means the membrane is broken and consequently the cell is dead.

There are 2 main types of dead cell marker dyes used:

1. DNA Staining Fluorochromes: Some DNA staining dyes cannot normally pass through the cell membrane due to their charged nature or may be kept out of cell by different membrane pumps. However, when the cell membrane is broken the dye will stain the entire nucleus of the cell.
2. Amine Staining Fluorochromes: These dyes stain proteins, again due to their charged nature they normally stain only a relatively small number of proteins on the surface of the cell. When the cell membrane is broken, they stain all proteins within the cell.

Example of using a DNA staining dye for cell viability assay.

The following is an example staining procedure for utilisation of DNA dyes such as Propidium Iodide (PI) for cell viability or 7-aminoactinomycin D (7-AAD).

This use of this method is for illustrative purposes only. Please ensure you refer to the specific product insert sheet of the product you are using.

1. PI is most commonly available as a powder, create a 1 mg per ml stock solution in water. 7 AAD is usually available as a liquid.
2. Stain cells for surface antigens first, do not fix, wash cells 1-2 times with PBS or a proprietary staining buffer
3. Resuspend cells in an appropriate volume of the staining buffer suitable for running on your instrument.
4. Add 5µL of Propidium Iodide stock solution or 1 test of 7-AAD staining solution per 100 µL of cells.
5. Incubate for 5-15 minutes on ice or at room temperature. Do not wash or fix cells after staining. Note: Propidium Iodide and 7-AAD must remain in the solution during acquisition.
6. Analyse samples by flow cytometry. Note: Cells should be analysed within 4 hours after the initial incubation period mainly due to the uptake of the dyes by living cells if left in the presence of propidium iodide or 7-AAD for prolonged periods. Store samples at 4-8°C and protect from light until ready for analysis.

DNA staining dyes for dead cell exclusion.

Important notes:

• Do not wash cells. The staining dye needs to be in equilibrium. Always keep dye in sample, even when running solutions.
• Run on cytometer within 1-2 hours of staining.
• You cannot fix post staining.

Example DNA dyes for cell viability assays.

The major excitation wavelengths are shown underscored. However, due to there being so much DNA present, other excitation wavelengths may be used to give a visible signal (this is shown in smaller font).

Amine staining dyes for the exclusion of dead cells.

Example Amine Reactive Dyes.

There are many companies which distribute amine viability dyes. The tables below show a selection of dyes from a few of those companies.

BioRad VivaFix Range

BioLegend Zombie Dyes

Ebio (ThermoFisher) “LIVE/DEAD Fixable” Range

Example of using an amine-based dye for cell viability assay.

The following is a general example of how to utilise fixable viability dyes

Use of this method is for illustrative purposes only. Please ensure you refer to the specific product insert sheet of the product you are using.

It is highly recommended that the user titrates the product for use in their own system using appropriate negative and positive controls.

Please note

A buffer without sodium azide, serum, or protein can be used in place of phosphate buffered saline.

Staining with fixable viability dyes can be done before or after antibody staining.

1. Prepare the viability dye stock solution as per the manufacturer’s instructions.
2. Thoroughly wash cells once in serum and azide free pbs solution to eliminate any protein then resuspend cells at around 1 million cells per ml.
3. Add the recommended amount of fixable viability stock solution.
4. Incubate the mixture for 30 min at room temperature.
5. Wash cells twice in phosphate buffered saline containing protein.
6. Fix cells in formaldehyde. Wash cells after fixation.
7. Resuspend cells in the appropriate flow analysis buffer. The viability staining is stable sample can be stored or diluted before use.
8. Run cells on your Flow Cytometer.

DNA staining vs Amine staining dyes as dead cell markers.

DNA Staining Dyes Advantages and Disadvantages

Disadvantages:

• Dye must be present in cell suspensions when running on a cytometer. No cell washes permitted as the dye will leach out of positive cells.
• Must be used on unfixed cells only.
• Must be washed thoroughly from the cytometer as it will stain future fixed samples. To clean run on system use; 1 wash of 1:10 bleach solution for 3 minutes followed by 3 washes of water.

Advantages:

• Fluorescence is very bright, especially from 4’,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), dead cells are very well separated from living cells.

• Shortly after staining living cells have zero fluorescence, so colour compensation of living cells into other cells is not necessary. Colour compensation of positive dead cells into other detectors is also not necessary, as they will be gated out of all subsequent plots. However, you will still need to compensate other colours that overlap into the detector used for dead cell signal detection for best gating separation.

Note with DAPI, PI, & 7AAD living cells can stain slightly after 60 minutes in dye. So may not be suitable for plate staining methods. DRAQ7 reportedly has no uptake in living cells over long periods, so could be a good candidate for plate-based assays

• DNA Dyes have no effect on living cells so can be used in functional assays and cell sorting
• DAPI and PI are very, very cheap, 20mg will last many years, 7 AAD and DRAQ 7 cost slightly more, but are still relatively cheap.

Amine Staining Dyes Advantages and Disadvantages

Disadvantages:

• Can be dim and the separation of dead cells from living may only be adequate. This can be further hindered by living cells also being weakly positive.
• Care is required in colour compensation;
  • Unfortunately living cells as well as dead cells can be stained. Compensation must be correct for the living cells.
  • The staining of positives can be weak – compensation must be optimised to enable the correct gating. 
  • A compensation control is needed; antibody capture beads cannot be used for this purpose, so dead cells are required and there may be only a few in the sample – making dead cells intentionally is more difficult than you think!
• Will require titration to decrease living cell fluorescence.
• Dyes bind to proteins so you will need to ascertain cell function after staining if sorting living cells.
• Costs not as high as antibodies but can still be significant.

Advantages:

• Are used as part of the staining procedure and can be fixed as the staining is permanent.
• Tends not to contaminate the cytometer system as they are blocked by proteins in the wash fluids you used to wash your cells during prep- no extra washing of the cytometer required.
• An unstained negative control not normally required, as in the stained sample both living and dead cells are stained.
• Costs not as high as antibodies but can still be significant.
• They are great for intra-cellular staining experiments.

We hope this short article will help you understand the importance of cell viability and the difference between amine and DNA dyes and when it is best to use each.