Fluorescent Cell Testing Measures – Assessing cell proliferation and cell health using flow cytometry and imaging platforms

Propidium Iodide (PI) is a cell impermeant DNA binding dye. In a population of cells, there are live cells and dead cells, and PI can be used to identify dead cells in a mixed population. In this case a healthy cell membrane will exclude the dye; the cell membrane forms an intact barrier to exclude the dye from getting into the cell. When a cell is dead, the cell membrane is compromised, and the PI dye is allowed inside the cell where it can bind to the DNA and become fluorescent, and thus identify the dead cells. This uses a log scale on your flow cytometer.
When cells are fixed, PI can be used to determine DNA content cell cycle. During fixation, the cell membrane loses its integrity and so PI may freely enter the cell to bind to nucleic acids. Because PI is not DNA-selective, remember to add RNase to your staining solution. This uses a linear scale readout on your flow cytometer.

Imaging of non-adherent cells can be difficult, mainly because they don’t image very well. Here are some things to consider:
• confocal imaging will help since non-adherent cells tend to be spherical
• if labeling intracellular structures, washes and optimization is really important since the internal cellular architecture is not as nicely spaced out like in adherent cells
• use of coated plates/dishes help the cells adhere a bit, helping to prevent movement
• keep the imaging media at a constant temperature, to help decrease liquid movement that may disturb the cells
• finally there are imaging cytometer systems that, although expensive, are very well suited to performing imaging with cells in suspension

There are a number of choices for evaluating the health of your cells using fluorescent imaging assays, from simple to complex. Understanding how healthy your cells are, and determining optimal timing for passage can improve productivity. A number of assays can help provide useful information.
•To evaluate viability, the use of a cell-impermeant DNA binding dye may be used, such as the Image-iT DEAD Green Viability stain where dead cells will fluoresce green. A dead cell stain can be combined with a marker of cell vitality that measures esterase activity, Calcein AM, as seen with the LIVE/DEAD Cell Imaging Kit where active cells fluoresce green and dead cells fluoresce red. Probably the most simple method uses ReadyProbes Dead Cell stains; these are ready-to-use reagents for fluorescent imaging that have been simplified for use (add 2 drops from the dropper bottle per mL of sample and image).
•Cell proliferation analyses can be helpful for assessing cell growth. A very useful method uses incorporation of a thymidine analog, EdU, for direct measurement of cells in S-phase using click chemistry. The Click-iT Plus EdU assays are fully compatible with fluorescent proteins.
•You may want to know if your cells are dying via apoptosis, the CellEvent Caspase 3/7 Green reagent is a no-wash assay compatible with labeling in media, the green fluorescence intensity increases with caspase activity.

DNA content cell cycle analysis requires careful optimization of every step in the process. Under ideal staining conditions, all cells with the same DNA content are expected to be uniform in staining. However in practice, variation can result from differences in sample prep, staining, methods of acquisition and anaylsis, along with performance of instrumentation. For a given experiment, each sample should contain the same number of cells, as sample to sample variation in cell number leads to significant differences in fluorescence signal. Having a single cell suspension is important is cell staining, as cell clumping or aggregation limits the accessibility of the dye to all cells equally. Methods to remove aggregation before staining include trituration and filtration. Methods used in analysis include as pulse-processing used to remove cell aggregates thru gating and software modeling used to eliminate cell aggregates and debris from the analysis. With hydrodynamic focusing cytometers, acquire in a low flow rate for the most precise results. During the acquisition, you may try waiting a few seconds to allow stabilization of each sample before recording.

Fixation has two functions. First, it preserves the cells by preventing lysis and autolytic degradation. Second, it makes the cells permeable and thus their DNA accessible to impermeant DNA-binding dyes.
Precipitating fixatives (methanol, ethanol, acetone) are preferred for single color cell cycle staining. Disadvantages include some epitpoes are destroyed and there is more cell aggregation.
Crosslinking fixitves (-aldehydes) can have a harmful effect on stoichiometry of DNA staining, but fewer epitpoes are destroyed and less cell aggregation is induced. Although inferior in terms of stabilization & preservation of low molecular weight constituents within the cell, they adequately stabilize undamaged DNA.
You will need to choose the best fixation method for your application. Or you may use a cell-permeant dye such as the Vybrant DyeCycle stains or UV-excited Hoechst 33342 for cell cycle in living cells.

There is another popular dye, CellTrace Violet, which uses violet excitation with blue emission. CellTrace Violet is fully compatible with GFP. CellTrace Violet and CellTrace CSFE are both amine-reactive succinimidyl ester compounds that covalently bind to proteins and give a bright homogenous fluorescence. When cells divide, the fluorescent label is distributed equally between daughter cells. As a result, each successive generation in a population of proliferating cells gives a reduction of fluorescence intensity, and generations can be identified.

Live cell imaging has transformed the way biologists study cells, proteins and a variety of processes and molecular interactions. Live cell imaging techniques allow the observation of internal structures and cellular processes in real time, and across time. Understanding cellular structures and dynamic processes can be critical in the study of cell biology. The observation of dynamic changes provides more insight into the processes of a cell, as compared to a snapshot provided by imaging studies of fixed cells. Since live cell imaging is less prone to experimental artifacts, it usually provides more reliable and relevant information than does fixed cell microscopy.
Live-cell imaging can be challenging. Labeling with fluorescent probes needs to be optimized for the particular assay readout, spectral compatibility and signal-to-noise level, and live-cell imaging of dynamic processes requires active observation over time. For reliable imaging results with live cells, it is recommended that the cells be healthy and maintained as closely as possible to physiological temperature, pH, oxygen tension, and other conditions. The use of an onstage incubator, an environmental chamber to enable control of temperature, humidity, and gases for time-lapse imaging of live cells under both physiological and non-physiological conditions, can be very useful in live cell imaging.

Phenol Red can cause problems with imaging, it is best to use a media that does not have phenol red in the formulation. One challenge with live cell fluorescence imaging is in imaging weak fluorophors without causing cell damage, photobleaching, or changes to cell health. A newer media from Gibco is FluoroBrite DMEM; it is a DMEM-based formulation with background fluorescence that is comparable to PBS and much lower than that from standard phenol red–free DMEM. FluoroBrite DMEM is designed to enhance the signal-to-noise ratio of fluorophors to enable detection of weak fluorescent events in an environment that promotes optimum cell health.

Some general causes of cell death include contamination from infections (yeast, bacteria, mycoplasma, viruses), chemicals (sterilizing solutions like ethanol or bleach), or issues with the cell line or methods of culturing.
To establish and successfully maintain cell cultures requires standardized approaches for media preparation, feeding, and passaging of the cells. Cultures should be examined regularly to check for signs of contamination and to determine if the culture needs feeding or passaging.
A quick look at the cell culture with basic brightfield microscopy can provide general information on gross contamination with microorganisms or if there is cross-contamination with cells from another culture.
pH drops with most infections of bacteria and yeast, and sometimes with fungal infections. Changes in pH can be measured with pHrodo AM indicators to look at cytoplasmic pH.
Many adherent cell cultures will stop proliferating once they become confluent, and some will die if they are left in a confluent state for too long. Suspension cells will exhaust their culture medium very quickly once the cell density becomes too high. Visualization of the cells with basic brightfield microscopy can help monitor confluency, however, testing of the cells to understand proliferation rates and changes in proliferation can provide very useful information on when it is best to passage a particular cell line. The Click-iT EdU assay can be sued, where the fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized.
The growth rate of cells that have been subcultured repeatedly may sometimes decrease unexpectedly, and there are several options for measuring viability and vitality of your cells. Dye Exclusion using an impermeant dye like DAPI can identify dead cells, while vitality can be measured with assays that include Calcein AM or C12-resazurin.
You may want to also investigate if your cells are undergoing oxidative stress, or apoptosis, and there are easy assays available for these with the CellROX reagents and CellEvent Caspase 3/7 reagent.

Often there is a delay between sample prep+staining before acquisition on the flow cytometer. Depending on the assay, the stained cells may be fixed and data acquired within 24 hours, but not all assays can be fixed in this manner. Some assays require the cells be fixed as part of the sample prep and staining, and usually you can acquire the data hours later. If you are performing a live cell assay that cannot be fixed, it is best to acquire the data as soon as possible; sometimes placing the labeled cells at 4º C may help. For live cell assays, I recommend looking at the data sheet from the manufacturer for guidance on compatibility with fixation and on storage before acquisition.

You can use fluorescent labels to identify dead cells, to identify live cells, or combine them both in a two parameter assay.

The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide or a dye from the STYOX series. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell where it will bind to the DNA and become fluorescent. The dead cells therefore will be positive and the live cells will be negative.

If you need to fix your cells, there is another option, using the LIVE/DEAD fixable stains, also known as amine reactive dyes. This also works using the principle of cell membrane integrity, where amines on the outside of a living cell with react with the amine-reactive dye and have a dim fluorescence, while a dead cell with a compromised membrane will allow the dye into the cell where it will react with amines throughout the cell resulting in a bright fluorescence. Unlike the DNA-binding dyes, the amine reactive dyes are fixable. You can label you cells with the LIVE/DEAD Fixable stain, and then fix the cells, and the distinction of live and dead cells will be maintained.

You may want to also look at a metabolic indicator, such as Calcein AM or C12-resazurin, so called vitality indicators. A metabolic label and a dead cell stain can be combined for a more complete look at cell health.

What you are testing for will determine the best method for detecting intercellular events. If your target of interest requires spatial resolution, imaging is the best platform to use. The cells will need fixation and permeabilization before labeling. The technical data sheet for the fluorescent product will list this out. If you are looking for a more quantitative approach with a population of cells, flow cytometry is a good choice with its capability for rich statistical information. The cells will need fixation and some assays will require fixation and permeabilization. Of course, live cells assays that use a cell-permeant dye to look at intercellular events do not require cell fixation techniques, for either imaging or flow cytometry platforms.