Troubleshooting for flow cytometry

Possible causes

Solution

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 positive single color control is set up correctly on flow cytometer, gated and compensated correctly to capture all the events

Compensation beads can be used

Insufficient antibody present for detection

Increase amount/concentration of antibody to optimise the working dilution

Intracellular target not accessible

• 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.
• Mild permeabilization buffers such as  0.1 % to 0.5% Saponin, Digitonin, and Tween 20 usually work well with cytoplasmic antigens (including the cytoplasmic face of the plasma membrane) and soluble nuclear antigens. Harsh permeabilization conditions such as 0.1 % to 1% Triton X-100 may be needed for some cytoskeletal and nuclear antigens.

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

Test using a fluorochrome with a lower molecular weight

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.

Ensure lasers on flow cytometer are aligned correctly by running flow check beads and adjusting alignment if necessary. If the lasers do not align correctly or if drift occurs, you may need to consider having the machine serviced.

Target protein not present/expressed at low level

• Ensure tissue/cell type expresses target protein and that it is present in a high enough amount to detect
• Verify through literature that the protein of interest is expressed in the cell type under investigation. When the target is present at low abundance or occurs in a rare cell population, incorporate a co-stain to help distinguish these cells or enrich the cell type of interest using a cell isolation kit before staining. Additionally, include known positive control samples to validate the assay and confirm accurate detection of the target protein.

Soluble/secreted target protein

A golgi-block step, such as with Brefeldin Aor Monensin, may improve the signal achieved for intracellular staining

Offset too high/gain too low

Use the positive control to set up the flow cytometer correctly again, using the offset to ensure the fluorescent signal from cells is not being cut off, and increase the gain to increase the signal (within reason – care should be taken)

Fluorochrome fluorescence has faded

Antibody may have been kept for too long or left out in the light

Fresh antibody will be required

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.

Use secondary antibody that was raised against the species in which the primary was raised (eg if the primary is raised in rabbit, use an anti-rabbit secondary)

Mismatch between dye brightness and antigen expression in multicolor experiments.

• Consult published data to determine the antigen density for your specific cell type.
• When designing your experiment, pair highly fluorescent dyes with antigens that exhibit low expression to ensure optimal signal detection and accurate results.
• Our flow cytometry panel builder can help you match fluorochromes with antigens based on antigen density.

Stimulation may be required for target protein expression.

Certain proteins are only expressed following stimulation. Experiment with various agents, concentrations, and treatment durations to achieve optimal expression. Examples: PMA, Ionomycin, LPS

Possible causes

Solution

Antibody concentration too high

High antibody concentration will give high, non-specific binding or very high intensity of fluorescence

Reduce the amount of antibody added to each sample

Excess antibody trapped

This can be a particular problem in intracellular staining where large fluorochrome molecules on the antibody can be trapped

Ensure adequate washing steps and include Tween or Triton in wash buffers

Inadequate blocking

Add 1% to 3% blocking agent with antibody as well as a blocking step

Gain set too high/offset too low

Use the positive control to set up the flow cytometer correctly again, using the offset to reduce background from small particles and reduce the gain to decrease the signal

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

Fc receptor blocking.

Fc receptors, present on myeloid cells, B cells and NK cells, should be blocked to prevent false positives.

Use an FC blocking reagent:

• ab324362
• ab324363

Excess dead cells are present in the sample.

• Dead cells can bind antibodies, leading to false-positive results. Use cell viability dyes to evaluate cell health.
• Prepare a fresh cell sample if the cell viability is very low.

Possible causes

Solution

More than one cell population present expressing target protein

Check expected expression levels from the cell types contained in the sample and ensure adequate cell separation if necessary

Cell doublets present

Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot

• Mix cells gently before staining and again before running on the cytometer using a pipette

• Cells can also be sieved or filtered to remove clumps (30 μl Nylon Mesh)

Possible causes

Solution

Cells lysed

Lysis can occur if samples are not fresh or prepared correctly

• Ensure cells in the sample have not lysed and broken up

• 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

Ensure sample is not contaminated

Possible causes

Solution

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

Run 1x10⁶ cells/mL

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 4°C during preparation

• Ensure you mix again before running

• In extreme cases, cells can be sieved or filtered to remove clumps (30 μL Nylon Mesh)

Possible causes

Solution

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

Dilute to between 1x10⁵ and 1x10⁶ cells/mL