Detailed troubleshooting techniques are provided here to help you resolve indirect staining flow cytometry issues. Though the troubleshooting guide below covers a multitude of problems encountered while performing your experiments, we do not expect it to be the exclusive solution to any problems. We do hope that you will find the information beneficial to you and useful as a reference guide in troubleshooting any problems you may encounter in indirect staining. If you ever need more assistance with your experiments, please contact us for additional help.
Click for the Protocol of Indirect Staining Flow Cytometry.
Contents
No Signal / Weak Fluorescence Intensity
Cause | Solution |
Insufficient detection antibody | Increase the concentration of detection antibody. |
Primary and secondary antibody are not compatible | Secondary antibody should be raised against the host species of the primary antibody. |
Antigen-antibody binding is sub-optimal |
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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. |
Antibody storage |
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Higher cell concentration | Adjust cell population to recommended density. |
Others |
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High Fluorescence Intensity
Cause | Solution |
High antibody concentration |
|
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 |
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Excess antibody |
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Non-Specific Staining
Cause | Solution |
High autofluorescence |
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Non-specific cells are targeted |
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Excess, unbound antibodies are present in the sample | Wash cells adequately after every antibody incubation step. |
Presence of dead cells |
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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 |
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Bacterial contamination |
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Presence of dead cells |
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Incorrect instrument settings for scatter |
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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 |
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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. |