A main advantage of flow cytometry over many other immunoassay techniques is that it allows researchers to study cells on an individual basis. Yet, the complex, multiparametric nature of flow cytometry can make resolving any issues difficult, especially when sample preparation, panel design, and instrument setup could all be to blame for any problems. Here, we’ve compiled some common flow cytometry complaints and suggested ways of addressing them. Although this is by no means an exhaustive list, we hope it will serve as a useful reference for troubleshooting your flow cytometry experiments.
A typical flow cytometry experiment begins with sample preparation, which must be carefully thought through to avoid compromising results. “Samples should ideally be processed as soon after collection as possible to help preserve cells in their natural state and maintain high viabilities,” notes Pia Jeggle, Ph.D., Global Product Manager, Flow Cytometry Instruments, at Miltenyi Biotec. "Where material will be analyzed within 48 hours of collection, keeping samples in a specialized cell or tissue storage solution at 4°C is recommended over freezing. However, in situations where freezing is unavoidable—like where material is collected from large cohort studies or during surgery—methods should be rigorously optimized to prevent undesirable effects such as cell death, altered cellular composition, or the induction of stress genes.”
Critically, the sample-preparation method should always be tailored to the cell type of interest; for example, isolating fragile neurons from brain tissue requires a very different approach than that used for isolating immune cells from blood.
Controls are essential to demonstrate reliable assay performance and validate experimental data. But, in addition to the usual positive and negative controls required for any biological assay, a well-designed flow cytometry experiment should also include several application-specific examples. “Unstained controls will help in determining light scatter and fluorescence parameters during instrument setup,” explains Mike Blundell, Product Manager for Flow Cytometry Reagents at Bio-Rad. “These should be complemented by a viability control to exclude dead cells, which bind antibodies non-specifically and have higher autofluorescence, as well as by controls for safeguarding the performance of multicolor panels.” Examples of the latter include single-stained compensation controls that are essential to remove overlapping fluorescence from neighboring channels, and fluorescence-minus-one controls, which are used for gate setting. “Provided you have good controls, identifying just ten cells can represent a significant result,” says Blundell. “In contrast, using poor controls may mean data collected from thousands of cells is meaningless.”
Although flow cytometry is distinct from other immunoassay techniques, the types of issues it presents can often be resolved using tried and trusted methods. For example, one way of reducing high background signal is to try increasing the number of protocol wash steps. Where problems are application-specific, solutions aimed at optimizing panel design or altering the instrument settings may be more effective. The following table lists some common flow cytometry complaints and suggests ways of addressing them.
Switch to using a different blocking reagent
Increase the duration of the blocking step
Titrate antibody reagents to determine optimal concentrations
Include positive and negative (unstained) controls to validate antibody performance
Increase the number of wash steps
Include Tween-20 or Triton-X in wash buffers to ensure unbound antibodies are not trapped inside the cells
Always pair dim fluorophores with abundant antigens, and vice versa
Reduce the laser power (where the instrument allows this) to decrease the signal intensity
Confirm the right instrument settings have been loaded prior to acquisition
Include suitable controls to optimize instrument settings for each fluorophore
Perform voltage titration with the aforementioned controls to determine the optimal setting for the detector
Ensure an appropriate positive control is used for instrument setup
Reduce the gain to lower the signal intensity
Try using a different fixative or reduce the fixation time/concentration
Confirm antibody reagents are validated to detect the target in the selected sample type and species, and are compatible with the fixation method (if applicable)
Check that antibodies are validated for flow cytometry
Switch to using antibodies with alternative epitopes
Increase the duration of antibody incubation steps
Replace fluorophore-conjugated antibodies with fresh reagents and follow the manufacturer’s instructions for use
Store fluorophores and stained samples away from light
Avoid using tandem dyes for experiments that will occur over a long time course
Refer to the literature to confirm that the chosen cell type expresses the antigen and determine whether some form of treatment is required to boost expression
Use freshly-isolated cells where possible, avoiding freeze-thawing
Always pair bright fluorophores with scarce antigens, and vice versa
Consider performing cell enrichment (e.g., via magnetic activated cell sorting/MACS)
Optimize the method used for cell permeabilization
Prevent intracellular analytes from being secreted by using a protein transport inhibitor such as brefeldin A
Perform all protocol steps on ice to avoid internalization of cell surface antigens
Choose low molecular weight fluorophores for detecting intracellular targets
Check that the excitation and emission properties of fluorophores are compatible with the flow cytometer’s lasers and detectors
Adjust the cell population to an appropriate density (1 x 106 cells/mL is usually recommended)
Check compensation controls are set up correctly to capture all the events
Confirm that the gating is correct (i.e., the cells within the negative gate are negative and the cells within the positive gate are positive)
Consider using compensation beads if you have weak signal to ensure good compensation
Check laser alignment using calibration beads
Make sure an appropriate positive control is used for instrument setup
Adjust the threshold to ensure the fluorescent signal is not being cut off
Increase the gain to increase the signal intensity
Consider using antibodies without an Fc receptor blocking requirement (e.g., REAfinity™ antibodies)
Include isotype controls to rule out non-specific antibody binding to Fc receptors, fluorophores, or other cellular components
Incorporate an Fc blocking step
Pass cells through a cell strainer to remove any clumps before acquiring
Keep cells at 4oC or on ice
Include viability dyes such as propidium iodide (PI) or 7-AAD to gate out dead cells
For resuspension, use a buffer that is free of Ca2+/Mg2+ and contains up to 2% protein
Consider adding DNase to samples
Avoid over-fixation
Include unstained controls to determine background autofluorescence levels
Pair cells that have naturally high autofluorescence (e.g., neutrophils) with bright fluorophores or fluorophores that emit in the red channel (e.g., APC)
Use sterile cell culture techniques
Store reagents and stained cells properly to prevent bacterial growth
Always use fresh buffers (e.g., for blocking and washing)
Wash the sample line to ensure the instrument is not contaminated from a previous sample
Optimize sample preparation
Avoid harsh vortexing or centrifugations, or freeze-thawing cycles
Do not store stained cells for extended periods prior to acquisition
Consider using dead cell removal reagents
Confirm RBC lysis is complete by checking samples under a microscope
Prepare fresh RBC lysis buffer
Increase the number of wash steps during RBC removal
Check the literature to ensure the correct activation approach has been used and that no accidental activation took place
Double-check the cell count (1 x 106 cells/mL is usually recommended)
Consider using an automated cell counter instead of a hemocytometer for counting
Ensure the cells are mixed thoroughly by gently pipetting the suspension
Unclog the flow cytometer by following the manufacturer’s instructions
Refer to the manufacturer’s instructions
Refer to the literature for information about target expression
Adapt the staining strategy to eliminate unwanted cell types from the analysis
The fluorescence intensity of the controls needs to be as high, or higher than, the sample staining in its dedicated channel
Use the automatic compensation function in software programs rather than manual compensation
Avoid using visual alignment and, if manual adjustment is required, use mean fluorescent intensity (MFI) alignment