Troubleshooting Flow Cytometry Experiments

Flow cytometry is fast, sensitive, and quantitative at the single-cell level, and remains one of the most widely used immunoassay techniques available today. However, drawing accurate conclusions from flow cytometry data means addressing any potential problems early on. Here, six flow cytometry experts share practical guidance for producing results you can trust.

Where other techniques fall short

“Flow cytometry allows researchers to analyze thousands of individual cells per second, giving it a major advantage where other techniques fall short,” explains Haley R. Pugsley, Ph.D., Manager and Senior Scientist, Amnis Flow Cytometry at Luminex. “For example, let’s say the cell population you are looking for has a frequency of just 1 cell out of 10,000. Detecting that population using manual microscopy methods seems like an impossible task, yet with flow cytometry you can find the target cells almost instantly. Or suppose you want to measure the activation of nuclear factor kappa B (NFκB), which is hallmarked by its movement from the cytoplasm to the nucleus. The two non-flow cytometry-based methods for measuring nuclear translocation are microscopy, which has limited throughput, and western blot, a bulk analysis technique that can lead to information being lost. With flow cytometry you can measure the phosphorylation of NFκB—which is required for its transition from the cytoplasm to the nucleus—and you can do so extremely rapidly for thousands of cells to increase your chances of identifying a rare population.”

Inherent challenges

Problems encountered by flow cytometrists can often be attributed to one of three main sources, the first of these being the need for a single-cell suspension. “For sample types such as blood, or non-adherent cell lines, generating a single-cell suspension is relatively straightforward,” reports Kivin Jacobsen, Senior Research Scientist at Immudex. “But solid tumors and tissues are more challenging, and optimization of the cell singularization method is critical for producing good flow cytometry data.”

Complications can also arise from the requirement to detect multiple markers simultaneously. “While flow cytometry can now be used for detecting upwards of 20 markers, this benefit makes experimental design more complex,” notes Emily Cartwright, Senior Product Marketing Specialist for Antibodies at Bio-Techne. “Factors to consider include the expression level of different antigens, whether several antigens are co-expressed on the same cell type, and the spectral properties of the fluorochromes.” Tools like Biocompare’s Flow Cytometry Panel Builder can be useful here.

Thirdly, protocol considerations become a challenge when the aim of the experiment is to detect both extracellular and intracellular targets. “Although some antibodies that are designed for use on live cells will also work well on cells that are fixed and permeabilized, others will only properly bind the target when added prior to the fixation step,” explains Christopher Manning, Associate Director for Flow Cytometry and High Content Analysis at Cell Signaling Technology. “Also, antibodies cannot be readily swapped between protocols without validation, so moving even trusted conjugates from one panel to another can involve work if the immunostaining protocol will vary between panels.” To improve understanding of protocol flexibility for commonly used phenotyping readouts, Cell Signaling Technology has compiled a Protocol Compatibility Table displaying this information.

According to Garret Guenther, Ph.D., Senior Global Support and Product Manager for Flow Cytometry at Agilent, other sources of problems for flow cytometry include incorrect instrument settings, unstable instrument alignment and/or fluidics, improper control samples, and incorrect data analysis. “Learning the fundamentals of flow cytometry along with application-specific details is key to producing quality data,” he says. “I recommend attending flow cytometry courses with lecture and lab components taught by leaders in the field, as well as identifying a flow cytometry expert at your institution or a nearby institution who can provide technical guidance during both experimental design and when collecting the data.”

Lastly, Pugsley highlights the intricacies of comparing data from different instruments since values like Mean Fluorescent Intensity (MFI) are in arbitrary units for each cytometer. “There is a move to have researchers present their data in calibrated units like Equivalent Reference Fluorophores (ERF) so that data from different instruments can be directly compared,” she says. “I think this is sensible to help standardize flow cytometry.”

Troubleshooting

Many flow cytometry challenges are easily overcome. The following are some common flow cytometry complaints, with suggestions of how to fix them, and you can also find further information in our Flow Cytometry Troubleshooting Guide.

The fluorescent signal is too high

When the fluorescent signal is too high, spectral spillover can decrease resolution. “Too high of a fluorescent signal could indicate incorrect instrument settings, specifically too much gain/voltage being applied to the detectors,” says Manning. “This can be addressed by reducing the gain/voltage to within the limits of the detector, or by decreasing the laser power if your flow cytometer allows this.”

If those adjustments are not sufficient, you may need to rethink fluorochrome selection. “Ideally, brighter fluorochromes should be reserved for detecting targets with low or unknown expression levels,” comments Melissa Thone, Field Application Scientist at Immudex. “In addition, titrating fluorescent reagents is important for ensuring that the separation is optimal and the signal is within the linear part of the scale.”

The fluorescent signal is too low

Incorrect instrument settings can also be responsible for the fluorescent signal being too low. However, low signal is more often due to a sub-optimal staining protocol. “There are numerous protocol strategies you can employ to boost a low fluorescent signal,” reports Cartwright. “These include keeping cells on ice to prevent surface antigens from being internalized, and testing different permeabilization methods to ensure cytoplasmic or nuclear targets are accessible to antibodies. You can also try adjusting activation protocols used to increase protein expression—remembering to use a Golgi inhibitor when detecting secreted proteins—and optimizing parameters such as the staining time and temperature.”

Thone also suggests considering reagents featuring multiple fluorochromes (e.g., Dextramer^® technology) to boost the signal of positive cells or employing a dual staining approach for targets that are of both low brightness and low frequency. “With dual staining, applying two different fluorochrome-labeled reagents with the same specificity permits visualization of the positive population on the diagonal in a flow plot, allowing for greater separation” she says. “For example, PE might be displayed on the X axis and APC on the Y axis.”

Samples have high background

Factors contributing to high background include non-specific antibody binding, cellular autofluorescence, and improper washing. “If fluorescence is high in cells where no signal is expected, check that the antibody is approved for flow cytometry and that it has been validated in the protocol you are using,” cautions Manning. “Another possible explanation for higher-than-expected signal is binding of the antibody by Fc gamma receptor proteins. If this is suspected, try using an Fc blocking reagent to minimize the interaction.”

“Running unstained controls is a quick and easy way to check for cellular autofluorescence,” adds Cartwright. “Also, using a viability dye to exclude dead cells and debris from analyses, as well as increasing the number of wash steps, will help to reduce unwanted background signal.”

The scatter profile is unusual

Guenther notes that an unusual scatter profile can be attributed to contamination, unstable fluidics, incorrect instruments settings, or poor instrument alignment. “Comparing the scatter profile to what is typical on an instrument will help you to better narrow down the cause,” he reports. “In general, high numbers of small events can indicate possible contamination, while a broad spread of events might suggest that a bubble is trapped within the fluidic path.”

“When I have an unusual scatter profile, I like to take a look at the cells to see what could be going on,” says Pugsley. “Because I routinely use an imaging flow cytometer, I can gate on the population and visually inspect the cells to see if there is something unexpected about them. For someone using a traditional flow cytometer, I suggest taking the sample to the microscope to see if there is anything out of the ordinary.”

Unexpected cell populations are observed

Finally, a list of common flow cytometry problems wouldn’t be complete without mentioning unexpected cell populations, which Guenther points out can sometimes be the most interesting. “Confirming whether unexpected results are real or artifacts hinges on using proper experimental controls,” he says. “In addition, excluding doublets from the analysis, filtering samples to remove clumps, and using the right instrument settings will all help to ensure that flow cytometry data are interpreted correctly.”

Whatever the aim of your flow cytometry experiment, reagent and instrument manufacturers are keen to help. Speaking with an experienced flow cytometrist before setting up your experiment is a fail-safe approach to maximize the quality of your data.