Flow Cytometry Troubleshooting: Common Problems and How to Fix Them

Author: Mike Blundell | Reviewer: Chloe Fenton

Flow cytometry troubleshooting helps identify common causes of poor staining, weak fluorescence, high background, and inconsistent results. If you are seeing no signal, weak staining, or nonspecific staining, start by checking antibody storage, fluorophore compatibility, antigen expression, controls, and instrument settings.

If something doesn’t work, check through the following guidelines to identify and resolve the problem. If you are still experiencing difficulties and have purchased your reagents from Bio-Rad, our Technical Services Team will be happy to offer further advice.

Quick Troubleshooting Reference

Problem Common Cause What to Check First
No staining Antibody, fluorophore, antigen, or instrument mismatch Storage, expiry, conjugation, antigen presence, laser/channel selection
Weak staining Low antibody concentration, low target expression, excessive cell density Antibody titration, target biology, sample density
Nonspecific staining Autofluorescence, Fc receptor binding, insufficient washing, secondary cross-reactivity Unstained controls, Fc blocking, wash steps, secondary selection
Unusual scatter profiles Sample condition or preparation issues Check cell viability, freshness, and buffer preparation
Unexpected staining Reagent interference or antigen localization issues Review sample prep, permeabilization, and reagents used
 

How to Troubleshoot a Flow Cytometry Experiment

  1. Confirm that antibodies and reagents have been stored correctly and are within expiry.
  2. Check that the target antigen is present in the sample and that the antibody is validated for the species used.
  3. Verify that the fluorophore, laser, and detection channel are compatible.
  4. Review controls, including unstained controls and any appropriate positive controls.
  5. Check sample preparation, washing steps, and cell density before repeating the experiment.

Common Flow Cytometry Problems and Solutions

Use the sections below to diagnose the most common flow cytometry issues. Each problem includes likely causes and practical checks to help you correct the issue quickly.

Problem Course of Action

Why am I seeing no staining in flow cytometry?

No staining
  1. Confirm that all antibodies have been stored correctly according to the manufacturer’s instructions.
  2. Confirm that commercial antibodies have not exceeded their date of expiration.
  3. Make sure that appropriate primary or secondary antibodies have been added.
  4. Make sure that antibody is conjugated to a fluorophore. If not, confirm that an appropriate fluorophore-conjugated secondary antibody is being used.
  5. Confirm that secondary antibody is active. Has it been used successfully with other primary antibodies?
  6. Make sure a secondary antibody that will recognize your primary antibody is being used.
  7. If the fluorophore used is phycoerythrin (PE) or allophycocyanin (APC) based, make sure that the product has not been frozen.
  8. Is the target antigen present on test tissue? Check literature for antigen expression and incorporate a positive control of known antigen expression alongside test material.
  9. Does antibody recognize antigen in test species? Check that antibody cross-reacts with species being used. Not all antibodies will react across species.
  10. Confirm that correct laser is being used to excite fluorophore, and that correct channel is being used to analyze emissions.
No staining with PE antibody but same FITC antibody gives good results.
  1. PE conjugate may have been frozen. If so, purchase another vial of antibody.
  2. Paraformaldehyde (PFA) may be a problem. Breakdown of PFA may release methanol, which will affect staining. Make up fresh PFA. Cells can be analyzed immediately without fixing.

Why am I seeing nonspecific staining in flow cytometry?

Nonspecific staining
  1. May be due to autofluorescence. Solution: check levels of autofluorescence by including only a tube of cells (that is, without any antibody) in your panel.
  2. Certain cells express low-affinity Fc receptors CD16/CD32, which bind whole antibodies via Fc region. Dilute antibody in Mouse Seroblock FcR (Bio-Rad product code BUF041A or B) for mouse cells, or Human Seroblock (Bio-Rad product code BUF070A or B) for human cells. 
  3. May be due to the secondary antibody. Select a secondary antibody that will not cross-react with target tissue.
  4. Make sure that sufficient washing steps have been included.
  5. Titrate test antibody carefully. Nonspecific staining may be reduced at lower antibody concentrations.

Why is my flow cytometry signal weak?

Weak staining
  1. May be due to overdilution of antibodies. Confirm that antibodies are used at the correct concentration by titrating antibodies before use.
  2. Weak staining in indirect staining systems may be due to prozoning effect, where highly concentrated antibodies may give weak results. Titrate antibodies carefully.
  3. May be due to an excessive number of cells. Adjust cell population to recommended density.
  4. May be due to the antigen expression. Check literature for expected levels of expression.
  5. If antigen expression is weak, select an antibody that is conjugated to a brighter fluorophore.
  6. May be seen if using a cross-reacting antibody rather than one specific for the target species.
  7. Optimize incubation time and temperature with either primary or secondary antibody.
Unusual scatter profiles
  1. Make sure that cells as fresh as possible are used. Profile may be showing dead cells and debris.
  2. Activation methods may affect scatter characteristics of cells.
  3. If you are using lysing solution, confirm that this is fresh and has been made up correctly.
Unexpected staining
  1. Some reagents may affect certain antigens and, therefore, may need reviewing. For example, EDTA will affect some platelet markers.
  2. Lysing solutions may affect certain antigens. Select a method that does not interfere with antigen detection.
  3. Some antigens are expressed intracellularly, therefore cell permeabilization methods may be required. Check manufacturer’s datasheet for correct permeabilization reagent.
 

Frequently Asked Questions

What causes no staining in flow cytometry?

No staining can be caused by incorrect antibody storage, expired reagents, missing primary or secondary antibodies, incompatible fluorophores, absent target antigen, lack of species reactivity, or incorrect laser and channel settings.

What causes weak staining in flow cytometry?

Weak staining may be caused by antibody overdilution, low antigen expression, a prozoning effect in indirect systems, or excessive cell density in the sample.

What causes nonspecific staining in flow cytometry?

Nonspecific staining may be caused by autofluorescence, Fc receptor binding, cross-reactive secondary antibodies, insufficient washing, or antibody concentrations that are too high.

How do I reduce background staining in flow cytometry?

Use appropriate controls, check autofluorescence, include sufficient wash steps, use Fc blocking where relevant, and titrate antibodies to reduce excess staining.

  
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