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Troubleshooting of Direct Staining Flow Cytometry

If something doesn’t work in your direct staining flow cytometry assay, please check through the following guide to resolve the problem. Though the troubleshooting tips provided here cover many different problems you may encounter in flow cytometry assay, we do not expect it to be the exclusive solution to any problems. We hope that you will find the information beneficial to you and useful as a reference guide in troubleshooting direct staining flow cytometry problems. If you ever need more assistance with your experiments, please contact us for additional help.

Click for the Protocol of Direct Staining Flow Cytometry.

Contents

No Signal / Weak Fluorescence Intensity

Cause Solution
Insufficient detection antibody Increase the concentration of detection antibody.
Sub-optimal antigen-antibody binding
  • Check the species specificity of the antibody.
  • Optimize the antibody incubation time and temperature.
  • Consider using biotinylated primary antibodies and an additional biotin-streptavidin step to amplify the signal.
The surface antigen is getting internalized Perform all steps at 4°C and use ice cold reagents.
Target protein not present or expressed at low level Ensure tissue or cell type expresses target protein and that it is present high enough to be detected.
Antibody storage
  • Ensure that all antibodies have been stored correctly.
  • Ensure that commercial antibodies have not exceeded their date of expiration.
Higher cell concentration Adjust cell population to recommended density.
Others
  • If antigen expression is weak, select an antibody that is conjugated to a brighter fluorochrome.
  • If the fluorochrome used is phycoerythrin or allophycocyanin-based, ensure that the product has not been frozen.
  • Ensure that correct laser is being used to excite fluorochrome, and that correct channel is being used to analyze emissions.

High Fluorescence Intensity

Cause Solution
High antibody concentration
  • Reduce the amount of antibody added to each sample. Too much antibody will result in high non-specific binding.
  • Use appropriate positive and negative controls.
Inadequate blocking Dilute antibodies in blocking solution.
A high expressing antigen is paired with bright fluorochrome Always pair strong antigens with dimmer fluorochromes such as FITC or Pacific Blue.
The fluorescent signal is under-compensated Use MFI alignment instead of visual comparison to compensate.

High Background

Cause Solution
The gain set is too high, the offset is too low
  • Use the positive control to set up the flow cytometer correctly again.
  • Use the offset to reduce background from small particles and reduce the gain to decrease the signal but do this within reason.
Excess antibody
  • Decrease antibody concentration.
  • Add a detergent to the wash buffers to ensure washing away of excess antibody.

Non-Specific Staining

Cause Solution
High autofluorescence
  • Always include an unstained control to subtract the auto-fluorescence signal.
  • For cells with naturally high auto-fluorescence, use fluorochromes that emit in the red channel where auto-fluorescence is minimal.
  • Use very bright fluorochromes for these cell types to amplify the signal above the auto-fluorescence level.
  • Avoid storing the cells in a fixative solution for long durations and analyze cells soon after staining if possible.
Non-specific cells are targeted
  • Include an isotype control to subtract any Fc binding signal.
  • Block the FcR on cells with Fc blockers, BSA or FBS prior to antibody incubation.
  • Additional washing steps.
Excess, unbound antibodies are present in the sample Wash cells adequately after every antibody incubation step.
Presence of dead cells
  • Always sieve the cells once before acquiring and sorting to remove any dead cell debris.
  • Include viability dyes like PI or 7-AAD to gate out any dead cells.
  • Use freshly isolated cells as opposed to frozen cells whenever possible.

Low Event Rate

Cause Solution
Low number of cells Optimal cell concentration for each sample should be around 1x106 cells/mL.
Cells clumped/blocking tubing Pipet the samples gently before staining and again before running the cytometer.

High Event Rate

Cause Solution
High number of cells Optimal cell concentration for each sample should be around 1x106 cells/mL.
Contamination Repeat the staining procedures.

Unusual Scatter Profiles

Cause Solution
The cells are lysed or damaged
  • Optimize sample preparation to avoid cell lysis.
  • Do not vortex or centrifuge cells at high speeds.
  • Use fresh buffers.
  • Avoid storing the stained cells for long durations.
Bacterial contamination
  • Store stained cells and antibodies properly to avoid bacterial growth. Bacteria will exhibit low levels of auto fluorescence.
  • Practice proper sterile cell culture techniques to prevent bacterial contamination.
Presence of dead cells
  • Always sieve the cells once before acquiring and sorting to remove any dead cell debris.
  • Use freshly isolated cells as opposed to frozen cells whenever possible.
Incorrect instrument settings for scatter
  • Ensure proper instrument settings are loaded prior to acquisition.
  • Use fresh, healthy cells to correctly set the FSC and SSC settings for that cell type.

Loss of Epitope

Cause Solution
Sample was not kept on ice Keep antibodies at 4°C to prevent loss of activity. This also prevents active phosphatases and proteases from altering the epitope of interest.

Two or More Cell Populations Are observed When There Should Be Only One Population

Cause Solution
Cell doublets are present in the sample
  • Mix the cells gently before the staining process and again before running them on the cytometer using a pipette.
  • Sieve or filter cells to remove clumps.
There is more than one cell population present which is expressing the target protein Check the expected expression levels from the cell types that are present in the sample and ensure adequate cell separation if necessary.