Troubleshooting | Flow Cytometry
Follow our flow cytometry troubleshooting guide to quickly identify and get solutions for common protocol issues.
First identify the problem with your flow cytometry test results from the options below:
Weak or No Signal
Signal not correctly compensated:
- Ensure that the positive single colour control is gated/compensated correctly on the flow cytometer to capture all events.
Cells were not permeabilized:
- Methanol and acetone fixation will permeabilize cells
- If using formaldehyde, permeabilize cells with 0.2% Triton X-100
Target protein expression is low:
- Perform proper treatment to induce expression of the target protein
- Run appropriate controls (ie. positive control, untreated control etc.)
- If the protein is present, but not abundantly, use an amplification step to maximize the signal
- Concentrate your protein lysates by isolating the cellular compartment containing your protein of interest (ie. mitochondria or cellular membrane)
Cell are over fixed:
- Reduced the duration/concentration of fixation. We recommend 1% formaldehyde for 15 min or less.
Fluorophore is dim:
- Pair a bright fluorophore with a low expressing protein
- Avoid over exposure of the slide to light sources for extended periods to avoid photobleaching. Always store fluorescent probes in the dark.
Gain is too low/Offset too high:
- Turn up the gain to ensure you are capturing any signal present
Primary and secondary antibodies are incompatible:
- The secondary antibody should be raised against the host species of the primary antibody. For example, if the primary was raised in mouse (ie. Mouse Anti-HSP70) use an anti-mouse secondary (ie. Goat Anti-Mouse).
Incorrect laser/filter set:
- Ensure your flow cytometer is equipped with the correct lasers and filter sets for the fluorophores you have chosen
Not enough primary antibody:
- Use a higher concentration of antibody
- Incubate longer
Antibody Storage issues:
- Freeze/thaw cycles are detrimental and can cause degradation. It is best to create aliquots of smaller amounts as soon as the product arrives at your location.
- Antibody was not stored as recommended. Unfortunately this might require a new vial to be used instead.
- If the secondary was not stored in the dark (when using immunofluorescence), a new vial will need to be used instead.
The protein is not present in the tissues being tested:
- Run a positive control
- If the protein is present, but not abundantly, use an amplification step to maximize the signal
- Increase the duration of incubation of the primary antibody with the sample
High Signal Intensity
Antibody Concentration is too high:
- Reduce the concentration of the primary and/or secondary antibody used
Insufficient washing:
- Proper washing of the tissue between steps is critical. Ensure you are following the protocol guidelines for wash steps.
Blocking is insufficient:
- Increase the blocking incubation period and consider changing the blocking agent.
Gain is too high/Offset is too low:
- Use the positive control to set optimal gain and offset levels that reduce background levels while maintaining a strong signal.
Two or more cell populations instead of one
More than one cell population expresses target protein:
- A second population of cells could be due inadequate cell separation. Check the expression levels of the cell types in the sample, and perform appropriate cell separation.
- Cell doublets can be removed through filtration
High side scatter
Cells lysed:
- Prepare fresh samples to avoid cell lysis to dead cells, debris or
- Ensure samples are not contaminated with bacteria by preparing fresh samples
Low event rate
Cells clumped/blocking tubing:
- Mix cells gently by pipetting gently before running and use a filtered tube when running the sample
- Prepare cells at 1×106 cells/ml, and mix well before running.
High event rate
High number of cells:
- Dilute cells to between 1×105 and 1×106 cells/ml.
Contamination:
- Prepare fresh samples and run experiment again.