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Flow Cytometry Troubleshooting Tips

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for flow cytometry.

Flow cytometry is a fluorescence-based assay that simultaneously determines multiple characteristics, such as cell population counts and protein abundance, from a single cell suspended in solution. It is a powerful tool for rapid, quantitative, and accurate determination of cellular characteristics and provides excellent interpretation against cell population heterogeneity. Based on our rich hands-on experience, Creative Biolabs has summarized common flow cytometry solutions to help you run your experiments smoothly. We could also offer mAb discovery services based on fluorescent activated cell sorting (FACS) technology in a timely and cost-effective manner.

No Signal/Weak Fluorescent Intensity

  1. Signal not correctly compensated

    2. Correctly set up on the flow cytometer to check for positive monochrome controls and correctly set the gate and compensation to capture all events.

  2. Detection of insufficient antibodies

    3. Increase the amount/concentration of antibodies.

  3. Intracellular target not accessible

    4. Check if the target protein is inside the cell. Internal staining is to be performed to ensure adequate permeability. To prevent internalization of cell surface proteins, everything must be done on ice or at 4°C with ice-cold reagents to stop all reactions.

    The addition of sodium azide will prevent the conditioning and internalization of surface antigens, which produces a loss of fluorescence intensity. For staining cell lines, trypsin can often induce internalization of cell surface proteins, especially cell surface molecules, which may require gentler isolation methods.

  4. Intracellular staining-fluorescent dye conjugate too large

    5. Fluorescent dyes used for intracellular staining experiments should have a low molecular weight. Fluorescent dyes with high molecular weights reduce antibody motility and may even interfere with antibody entry into the cell.

  5. Lasers not aligned

    6. Run the flow check beads to ensure that the lasers on the flow cytometer are properly aligned, and adjust the alignment if necessary. If the laser is not properly aligned or is drifting, it may be necessary to consider servicing the machine.

  6. Target protein absent/low level expression

    7. Ensure that the tissue/cell type expresses the target protein at a level sufficient for detection.

  7. Soluble/secreted target protein

    8. Is the target protein soluble and secreted from the cell? The target protein needs to bind to the cell membrane or cytoplasm to be easily detected by flow cytometry. A Golgi blocking step (e.g., using Brefeldin A) improves the signal for intracellular staining.

  8. Offset too high/gain too low

    9. Set up the flow cytometer correctly again using a positive control, use an offset to ensure that the fluorescent signal from the cells is not cut off, and increase the gain to increase the signal.

  9. Fluorochrome fluorescence has faded.

    10. Antibodies may have been stored too long or exposed to light. Fresh antibodies are required.

  10. Primary and secondary antibodies are incompatible.

    11. Use a secondary antibody directed against the species in which the primary antibody was produced (e.g., if the primary antibody was produced in rabbit, use an anti-rabbit secondary antibody.) (For antibodies that are not commercially available, we recommend our antibody customization service.)

High Fluorescence Intensity

  1. Antibody concentration too high

    2. This would produce high, nonspecific binding or very high fluorescence intensity. Reduce the amount of antibody added to each sample.

  2. Excess antibody trapped

    3. This may be a particular problem in intracellular staining, where large fluorochrome molecules on antibodies can be trapped. Ensure that there are adequate washing steps and include intermediates or triton in the washing buffer.

  3. Inadequate blocking

    4. Blocking agents and antibodies of 1% to 3% and blocking

High Background/High Percentage of Positive Cells

Gain set too high/offset too low Positive controls are used to reset the flow cytometer correctly, offsets were used to reduce the background from small particles, and gain was reduced to reduce the signal.
Excess antibody Decrease the antibody concentration. Detergent can also be added to the wash buffers to ensure any excess antibody is washed away.

Two or More Cell Populations Observed When There Should Be Just One

There was more than one cell population expressing the target protein. Check expected expression levels of the cell types contained in the sample and ensure adequate cell separation if necessary.
Cell doublets present Doublets of the cells will be shown as the second cell population on the plot, with approximately twice the fluorescence intensity. Cells were mixed gently before staining and again before running on the cytometer using a pipette. Cells could also be screened or filtered to remove clumps (30 μl nylon mesh).

High Side Scatter Background (from Small Particles)

Cells lysed Ensure that the cells in the sample are not lysed and fragmented. Samples should be fresh and properly prepared. Do not centrifuge the cells at high speeds or swirl them too vigorously.
Bacterial contamination Ensure that the sample is free from contamination. Bacteria autofluorescence at low levels. This will also have a high event rate.

Low Event Rate

Low number of cells/ml Run 1x106 cells/ml. Make sure the cells are well mixed gently.
Cells clumped, blocking tubing Before staining, homologous single-cell suspensions are ensured by gentle pipetting several times. Make sure to mix again before running. In extreme cases, cells can be screened or filtered to remove clumps (30 μl nylon mesh).

High event rate

High number of cells Dilute to between 1x105 and 1x106 cells/ml.

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