Troubleshooting for flow cytometry
Problems, causes, and solutions for issues in flow cytometry.
No signal/weak fluorescence intensity
Signal not correctly compensated
Within a flow cytometer, excitation and emission wavelengths are selected by bandpass filters. To correct for spectral overlap, fluorescence should be compensated so that what is detected comes from the fluorochrome being measured
- Check if target protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent internalisation of cell surface proteins, everything must be done on ice or at 4°C, with ice cold reagents, to stop all reactions.
- Adding sodium azide will prevent the modulation and internalization of surface antigens which can produce a loss of fluorescence intensity.
- For staining of cell lines, trypsin can often induce internalisation of cell surface proteins, particularly cell surface molecules, and more gentle detachment methods may be required.
Intracellular staining - fluorochrome conjugate too large
Fluorochromes for intracellular staining experiments should have a low molecular weight. Fluorochromes with large molecular weights can reduce antibody motility and its ability to enter the cell
Lasers not aligned
Within benchtop flow cytometers no operator adjustment is needed, however steam-in-air flow cytometers may need to have lasers adjusted to ensure it focuses on your sample core. This requires laser safety training and in most cases should be left to service engineers but if your alignement issues are consistent it may be best to have the machine serviced.
Soluble/secreted target protein
The target protein needs to be soluble and secreted from the cell (membrane bound or cytoplasmic) to be detected easily by flow cytometry
Fluorochrome fluorescence has faded
Antibody may have been kept for too long or left out in the light
The primary antibody and the secondary antibody are not compatible
Secondary antibodies should be carefully matched to your primary antibody across different characteristics. If you're struggling to find the right secondary antibody, read our guide on selecting a secondary antibody.
High fluorescence intensity
Antibody concentration too high
High antibody concentration will give high, non-specific binding or very high intensity of fluorescence
Excess antibody trapped
This can be a particular problem in intracellular staining where large fluorochrome molecules on the antibody can be trapped
High background/high percentage of positive cells
Excess antibody
This can be caused by high antibody concentration or excess antibody remaining
- Decrease antibody concentration
- Detergent can be added to the wash buffers to ensure any excess antibody is washed away
Two or more cell populations observed when there should be one
Cell doublets present
Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot
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Mix cells gently before staining and again before running on the cytometer using a pipette
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Cells can also be sieved or filtered to remove clumps (30 μl Nylon Mesh)
High side scatter background (from small particles)
Cells lysed
Lysis can occur if samples are not fresh or prepared correctly
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Ensure cells in the sample have not lysed and broken up
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Do not centrifuge cells at a high rotor speed or vortex too violently
Bacterial contamination
Bacteria auto fluoresce at a low level; this will also give a high event rate
Low event rate
Low number of cells/mL
Cell loss can occur across different stages in your protocol including preparation and permeabilization/fixation so always be sure to handle cells carefully
Cells clumped, blocking tubing
Clumps of cells block the stream of single cells required for flow cytometry, which can cause issues for your experiment and the next user in your lab. Necrosis and apoptosis can be kept to a minimum during prepartion if you keep your wash and buffers at 4oC during preparation
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Ensure you mix again before running
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In extreme cases, cells can be sieved or filtered to remove clumps (30 μL Nylon Mesh)
High event rate
High number of cells
Through overcaution or giving yourself too much room for error you may end up with a higher number of cells in your sample than needed. It's easy to dilute these to the necessary level