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Practical Advice to Set up and Troubleshoot your Flow Cytometry
Experiment for Immune Monitoring
Flow cytometry is a powerful technique to simultaneously analyze multiple traits of individual cells in a complex cell sample. Depending on the reagents used, such traits include intracellular and surface protein expression, cytokine production, and antigen-specificity.
Several factors influence the success of a flow cytometry experiment. Troubleshooting, therefore, can be complicated. However, keeping in mind a few important concepts during the setup of your experiment can save you time in the long run. In our experience, taking the time to carefully prepare samples, design your panel, and optimize staining with each reagent provides the best assay results from the start.
Here we explore those concepts to give you a leg up in setting up your experiment. If you already have results that appear suboptimal or unexpected, you’ll find links to troubleshooting recommendations in the following table.
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Problem |
Possible cause |
What to do |
More information |
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High fluorescence signal |
Suboptimal reagent-to-target ratio |
Titrate each reagent in a panel |
|
|
Low fluorescent signal |
Suboptimal reagent-to-target ratio Low avidity of target cell to reagent |
Pair bright fluorochromes to target molecules of low expression and vice versa Titrate each reagent in a panel Use multimer reagents |
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|
High background signal |
Low-quality samples Non-specific binding |
Focus your analysis on live cells Vary incubation time Wash away unbound reagent |
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|
Unusual scatter profile |
Inappropriate instrument settings Compromised sample |
Check your instrument settings; handle samples with care during staining |
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Unexpected cell populations |
Reagent interacts with non-target cells |
Reassess your gating strategy to exclude non-target cells |
Not All Samples are Alike
The nature of your sample plays a key role in the success of flow cytometry, especially in achieving specificity. Low-quality samples and the presence of dead cells can cause significant background interference. Therefore, include a viability stain in your panel design and focus on live cells. Knowledge about the tissue type obtained from the donor can also be essential, especially in the case of antigen-specific T cells. Furthermore, while antigen-specific cells are commonly characterized from blood samples where single-cell suspensions for analyses are not a problem, the appropriate dissociation of solid tumors or tissues to single cells is critical for good flow cytometry results.
“Include a viability stain in your panel design and focus on live cells.”
Even with high-quality samples, non-specific binding can generate background noise. Varying the incubation time can reduce that interference. Other times, a blocking reagent may be needed to prevent non-specific molecular interactions. For example, Fc block will prevent the non-specific binding of the constant region of antibodies to the Fc receptors of monocytes, macrophages, B cells and Dendritic cells. Finally, thoroughly washing the stained sample can alleviate background issues by removing unbound reagents.
Panel Design: Align Sample and Antibodies
Choosing the right antibodies to answer a research question and then optimizing your detection with the right fluorochromes is an involved process. The difficulty compounds in the design of larger panels or complex phenotyping schemes. Done incorrectly, a panel generates high data spreading, with the consequence of low resolution to tease apart cell populations. That low resolution is particularly problematic for examining antigen-specific immune cells. Given their low frequency in a sample, a suboptimal panel can mask their presence.
Select the specificities of your antibody panel to define a logical gating path that will reveal the antigen-specific immune cells of interest. Defining those specificities requires in-depth knowledge of immunology and the protein expression pattern of the cell types involved. Selecting inappropriate antibody panels leads to incorrect gating and wrong conclusions.
“Define a logical gating path that will reveal the antigen-specific immune cells of interest.”
Low avidity of a reagent to antigen-specific T cells can result in low fluorescence, making detection challenging. In these cases, using reagents with multiple fluorochromes – such as Dextramer® reagents, that incorporate multiple fluorochromes into each dextran backbone – can increase the signal of positive cells. Dual staining can also be an effective strategy to visualize target cells that are of low frequency and generate low brightness. In this approach, target cells are visualized with two differently labeled reagents with the same specificity. The positive cell population appears on the diagonal in a flow plot.
Signal-to-Noise Ratio: Optimal Staining
Choose the fluorochrome used for detection based on the expression density expected for the cells of interest. For example, T cells express CD3 and CD8 at far higher density than CD4. Thus, less bright fluorochromes suffice for their detection. Conversely, bright fluorochromes should be reserved to detect molecules of lower or uncertain expression levels. Having chosen a sensible fluorochrome, determine the optimal staining for detection by titrating the reagent. The goal is to achieve a consistent signal without an increase in background or reach an optimal signal to noise ratio.
“High avidity reagents that incorporate multiple fluorochromes can increase the signal of positive cells.”
Low avidity of a reagent to antigen-specific T cells can result in low fluorescence, making detection challenging. In these cases, using reagents with multiple fluorochromes – such as Dextramer® reagents, that incorporate multiple fluorochromes into each dextran backbone – can increase the signal of positive cells. Dual staining can also be an effective strategy to visualize target cells that are of low frequency and generate low brightness. In this approach, target cells are visualized with two differently labeled reagents with the same specificity. The positive cell population appears on the diagonal in a flow plot.
Further Help with Troubleshooting Flow Cytometry
Protocols and Spec Sheets
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MHC Dextramer® Reagents
Sensitively quantify target immune cells by flow cytometry with a strong signal-to-noise ratio.