Troubleshooting of Intracellular Staining Flow Cytometry

The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the intracellular staining flow cytometry experiments. Though the troubleshooting guide presented here covers many different problems encountered while performing your flow cytometry experiments, 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. If you ever need more assistance with your experiments, please contact us for additional help.

Click for the Protocol of Intracellular Staining Flow Cytometry.

Contents

Problem 1: No Signal / Weak Fluorescence Intensity
Problem 2: High Fluorescence Intensity
Problem 3: High Background
Problem 4: Non-Specific Staining
Problem 5: Low Event Rate
Problem 6: High Event Rate
Problem 7: Unusual Scatter Profiles
Problem 8: Loss of Epitope
Problem 9: Two or More Cell Populations Are observed When There Should Be Only One Population

No Signal / Weak Fluorescence Intensity

Cause	Solution
Intracellular target not accessible	Check if the target protein is intracellular. Check antigen expression and incorporate a positive control of known antigen expression alongside test material. Optimize cell permeabilization protocols.
Insufficient detection antibody	Increase the concentration of detection antibody.
Intracellular staining-fluorochrome conjugates too large	Fluorochromes with large molecular weight can reduce antibody motility and possibly halt its entry into the cell.
Primary and secondary antibody are not compatible	Secondary antibody should be raised against the host species of the primary 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 intracellular antigen is getting secreted	Try using a Golgi blocker such as Brefeldin A.
The surface antigen is getting internalized	Perform all protocol 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.
The fluorescence on stained cells has bleached	Acquire the cells immediately after staining. Add a fixative (like paraformaldehyde) to the samples if storing for an extended duration.
Antibody storage	Ensure that all antibodies have been stored correctly. Ensure that commercial antibodies have not exceeded their date of expiration.
Prozoning effect	Weak staining in indirect staining systems may be due to prozoning effect, where highly concentrated antibodies may give weak results.
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.
Excess antibodies are trapped in the cells in the case of intracellular staining	Wash the cells adequately after each antibody incubation step and include Tween or Triton X in wash buffers.
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 Fc receptors on cells with Fc blockers, BSA or FBS prior to antibody incubation. Additional washing steps. Include a secondary antibody-conjugate control to subtract any non-specific signal from the conjugate. Select a secondary antibody that will not cross-react with target tissue.
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
Excessive paraformaldehyde	Paraformaldehyde can release methanol in its breakdown, which may affect the staining. Make sure to use only 1% paraformaldehyde.
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.
Sample fixed too long	Optimize fixation protocol. Fixing the samples for too long may cause damage to cells. Most cells only need to be fixed for less than 15 minutes.

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.