Flow Cytometry Troubleshooting

Flow cytometry is a technology used to rapidly analyze cells or particles in a buffered salt-based solution. Flow cytometry uses one, or multiple, lasers to determine the visible light scatter given off by each cell. This light scatter is measured in two specific directions, forward and sideways, which provide information on the relative size of the cell and the internal complexity or granularity of the cell. Today, flow cytometers with multiple lasers are most common, and some instruments are even equipped for a specific purpose (e.g., coupled to a mass spectrometer). The latest generations of clinical flow cytometers include increasingly advanced scanning abilities, and can analyze cells at rates above 20,000 cells/second.

Prior to flow cytometry, samples must be stained or probed to allow for detection, which can be done in a number of ways. For example, cells can be transfected with recombinant fluorescent proteins like green fluorescent protein (GFP), stained with fluorescent dyes like propidium iodide (PI), or conjugated with fluorescent antibodies like CD3 or FITC. The availability and diversity of fluorochromes has also increased over the years, which in turn increased the number and type of analysis parameters available to detect. New recombinant fluorescent proteins have evolved from GFP into many different variants, like mCherry, mBanana, mOrange, and mNeptune.

The use of tandem dyes and polymer dyes has also helped increase the capacity of fluorochromes capable of conjugating to monoclonal antibodies. Data analysis techniques have also advanced from the traditional two parameter histogram (dot plot) gating methods to complex cluster data analysis, able to provide high-dimensional data.

Assaywise Letters:
Fundamentals of Flow Cytometry A practical guide for use of PE and APC in flow cytometry	Practical Guide for Live Cell Cycle Analysis in Flow Cytometry Therapeutic Monoclonal Antibody Fluorescent Labeling Tools for Imaging and Flow Cytometry

Common Issues and Solutions in Flow Cytometry

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Often, flow cytometry related issues are not apparent until after the data after the sample has already been tested. Some common issues, troubleshooting tips, and possible solutions are listed below.

Common Issue	Possible Reason/Troubleshooting Tips
Low fluorescence signal	Is the signal correctly compensated for? Ensure the positive single color control is set correctly on the instrument, gated, and compensated for to capture all the events. Is the amount of antibody sufficient for detection? Try increasing the amount and/or concentration of antibodies and retest. If not present at all, ensure the tissue and/or cell type expresses the target protein. Ensure the tissue and/or cell type is present in a high enough concentration (around 1x10e6 cells). If the target molecule is scarce, a bright fluorophore may be needed. If the antigen is intracellular, cells require fixing and permeabilizing for antibodies to access epitopes. Make sure that the fluorophores are matched with the flow cytometer lasers and detectors, and are protected from light to prevent photobleaching.
High background noise present	Is the gain set too high, or the offset too low? Adjusting the positive control offset will reduce the background, and reducing the gain will decrease the signal. High background can also result from inadequate blocking, washing, or antibody concentration. High background may be due to autofluorescence, to ensure cells are not over-fixed to minimize the presence of dead cells. Use red fluorophores for detecting highly autofluorescent cell types, like neutrophils. Keep samples on ice, avoid freeze-thawing, and include a viability dye to prevent/minimize dead cells. Are you detecting non-specific cell types? Use well-validated antibodies where possible, and if necessary, introduce an Fc blocking step into the experiment.
Fluorescence intensity is too high	Is the antibody concentration too high, leading to non-specific binding? Reduce the antibody concentration, and retest. Are instrument settings correct? Try decreasing the laser power, reducing the gain, or optimizing the photomultiplier tube (PMT) voltages. Modifications to the blocking step may be needed, e.g., switch to a different blocking solution, or increase the blocking time. Modification to washing steps may be needed, should excess antibody be trapped within the sample. Try increasing washing steps, or include a low concentration of tween, triton, or similar detergent into the buffer. Ensure that extremely abundant antigens are paired with dimmer fluorophores.
Intracellular target does not appear during analysis	Is the target protein intracellular? For internal staining, ensure adequate permeabilization. Ensure all reagents and steps are performed on ice. Gentler detachment methods may be required, and may require the addition of sodium azide which will prevent the internalization of surface antigens. When staining cell lines, trypsin may induce internalization of cell surface proteins.
Fluorochrome conjugate is too large	These fluorochromes should have a low molecular weight, as larger ones can reduce antibody motility and prevent entry into the cell.
The target protein is soluble or secreted	Target proteins should be membrane bound or cytoplasmic, not soluble or secreted from the cell. Protein transport inhibitors, such as with Brefeldin A, can be used to improve the signal.
Lasers not aligned	Ensure lasers are aligned correctly by running flow check beads and making adjustments, if necessary. This type of maintenance should be performed daily, prior to use.
Fluorochrome fluorescence appears faded	Antibodies may have been kept for too long or left out in the light. Reprepare the sample with fresh antibody and retest. Where applicable, ensure that the primary and secondary antibodies are compatible. Make sure that the secondary antibody was raised against the same species as the primary antibody.
Multiple cell populations are present when there should only be just one	Generally, more than one cell type is present that expresses the same target protein. Ensure adequate cell separation techniques are taken before retesting. The staining strategy may also need to be altered to accurately identify the population of interest. This may also be a result from antibodies binding non-specifically to dead cells.
Cell doublets are present	Doublets present as a second cell population at around twice the fluorescence intensity. Mix cells gently before staining, and prior to running through the cytometer.
High side scatter background is seen from small particles	Ensure cells have not lysed, or broken. Do not centrifuge cells at a high speed or vortex aggressively.
Autofluorescence or scatter properties are unusual	Ensure the sample is not contaminated. Bacteria tend to autofluoresce at low levels, and can emit a high event rate.
Low event rate	Ensure cells are thoroughly, gently, mixed and the starting population is >1x106 cells/ml.
Cells are clumping	Pipet gently before staining to ensure a homologous cell suspension. If necessary, cells can be sieved or filtered to remove clumps using a 30 μl nylon mesh.
Abnormal Event Rates	The event rate should be high enough for results to be statistically relevant, but not so high to compromise sample quality. Ensure the cell sample is between 1x10e5 and 1x10e6 cells/ml, and retest. If the event rate is too low, the flow cytometer could be clogged. If the even rate is too high, the sample may be contaminated with a large presence of cell debris.

Useful Tools

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Tools:
Flow Cytometry Panel Builder Spectral Flow Panel Builder Fluorescence Spectrum Viewer	Protein Concentration Calculator Detergents Data

Example Flow Cytometry Panels

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Further Reading

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Assaywise Letters:
Multicolor Labeling of Dead Cells for Flow Cytometric Analysis Multicolor Live or Dead™ Viability Assays for Flow Cytometry T Lymphocyte Immunophenotyping Using Multicolor Flow Cytometry	Comprehensive Guide to Cell Health Assays in Flow Cytometry: A Multidimensional Exploration Fluorescent Detection of Cell Senescence For Flow Cytometry and Imaging Applications

Resources

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Flow Cytometry Troubleshooting Guide
Technical issues: flow cytometry and rare event analysis
Flow Cytometry: An Overview