Flow cytometry training

Part 4: troubleshooting

---

Welcome to our training series on flow cytometry. We begin with essentials like getting to know your cytometer, before taking you through some of the finer points and ending with how to get the most from this powerful technique.

Welcome to the final part of flow cytometry training. Here we’ll share with you troubleshooting tips for different issues you’re likely to encounter in flow cytometry.

Part 4 overview

4.1 Troubleshooting video - no signal/high background
4.2 Compensation, spectral overlap, and calculation of spillover
4.3 Solving other issues encountered
4.4 FAQs

4.1 Troubleshooting video - no signal/high background

The main issues encountered with staining are either no signal or high background. The short video below will explain how to resolve these issues.

4.2 Compensation, spectral overlap, and calculation of spillover

In this video, we discuss how to carry out compensation, specifically how to calculate the spectral overlap of spillover, and how compensation is related to stability.

4.3 Solving other issues encountered

We have already discussed what to do if you experience no signal or high background issues; however, you may encounter other issues such as:

1. No signal/weak fluorescence intensity

Signal not correctly compensated
Check that the positive single color control is set up correctly on the flow cytometer, gated, and compensated correctly to make sure you capture all the events.

Insufficient antibody present for detection
Increase the amount/concentration of the antibody.

Intracellular target not accessible
Check if the target protein is intracellular or if the antibody epitope for a membrane protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent the internalization of cell surface proteins, all protocol steps must be performed on ice or at 4°C, using ice-cold reagents, to stop all cell reactions.

Adding sodium azide to your experimental reagents will prevent the modulation and internalization of surface antigens, which can produce a loss of fluorescence intensity. For staining of adherent cell lines, trypsin can often induce internalization of cell surface proteins, and more gentle detachment methods may be required.

Intracellular staining – fluorochrome conjugate too large
Fluorochromes for intracellular staining experiments should have a low molecular weight. Large molecular weight fluorochromes can reduce antibody motility and prevent the antibody from entering the cell to label your protein of interest.

Lasers not aligned
Ensure the lasers on the flow cytometer are aligned correctly by running flow check beads and adjusting the alignment if necessary. If the lasers do not align correctly or if drift occurs, you may need to consider having the machine serviced.

Target protein not present/expressed at a low level
Ensure the tissue/cell type you are analyzing expresses the target protein and that it is present in a high enough amount to detect.

Soluble/secreted target protein
Is the target protein soluble and secreted from the cell? Your target needs to be membrane-bound or cytoplasmic to be detected easily by flow cytometry. A Golgi-block step, such as with Brefeldin A, may improve the signal achieved for intracellular staining.

Offset too high/gain too low
Include a positive control to set up the flow cytometer correctly, using the offset to ensure the fluorescent signal from cells is not being cut-off, and increase the gain to increase the signal (within reason – care should be taken).

Fluorochrome fluorescence has faded
An antibody may have been kept for too long or left out in the light. The fresh antibody will be required.

The primary antibody and the secondary antibody are not compatible
Use a secondary antibody that was raised against the species in which the primary was generated (eg primary is raised in rabbit, use anti-rabbit secondary). Alternatively, to overcome the species cross-reactivity issues, you can use directly conjugated primary antibodies validated for flow cytometry, or add a desirable fluorochrome to your antibody of choice using a conjugation kit.

2. High fluorescence intensity

Antibody concentration too high
This will result in high, non-specific binding or very high fluorescence intensity. Reduce the amount of antibody added to each sample.

Excess antibody trapped
This can be a particular problem in intracellular staining, where large fluorochrome molecules on the antibody can be trapped within the cells. Ensure adequate washing steps and include tween or triton in wash buffers to maintain cell permeabilization.

Inadequate blocking
Add 1–3% blocking agent into your antibody mix, as well as a blocking step.

3. High background/high percentage of positive cells

Gain set too high/offset too low
Use the positive control to set up the flow cytometer correctly, using the offset to reduce background signal from small particles and reduce the gain to decrease the signal.

Excess antibody
Decrease antibody concentration. Detergent can also be added to the wash buffers to ensure any excess antibody is washed away.

4. Two or more cell populations observed when there should be just one

More than one cell population present expressing target protein
Check the expected protein expression levels in the cell types contained in the sample and ensure adequate cell separation if necessary.

Cell doublets present
Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. Mix the cells gently using a pipette before staining and again before running on the cytometer. Cells can also be sieved or filtered to remove clumps (30 μl Nylon Mesh).

5. High side scatter background (from small particles)

Cells lysed
Ensure cells in the sample have not been lysed and broken up. Samples should be fresh and prepared correctly. Do not centrifuge cells at a high rotor speed or vortex too violently.

Bacterial contamination
Ensure the sample is not contaminated. Bacteria will autofluoresce at a low level and result in a high event rate.

6. Low event rate

Low number of cells/ml
Run 1x10⁶ cells/ml. Ensure cells are mixed well (but gently).

Cells clumped, blocking the tubing
Ensure a homologous single-cell suspension by gently pipetting several times before staining. Ensure you mix again before running. In extreme cases, cells can be sieved or filtered to remove clumps (30 μl Nylon Mesh).

7. High event rate

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

4.4. Flow cytometry FAQs

Do the samples need to be fixed? Do I fix before or after staining?

Any samples, which contain potentially biohazardous material, should be fixed. We would recommend adding 1–2% paraformaldehyde to the sample once staining is completed. The samples should then be kept at 4°C in the dark and analyzed within 24 hours.

For intracellular staining, the cells are fixed and permeabilized together through the procedure. See our intracellular staining guide for more details.

Do the cells need to be permeabilized?

We recommend checking the datasheet for further details. If the target protein you are detecting is a membrane protein, then, most likely, you won’t require a permeabilization step. On very rare occasions, the antibody epitope may be in the cytoplasmic domain of the membrane protein. In this case, you may need to permeabilize with a gentle permeabilization agent, such as saponin. Check the data available on the datasheet (or Abreviews and images) to see if other researchers have required a permeabilization step. All cytoplasmic and nuclear target proteins will require cell permeabilization for antibody detection. For the detailed protocol, check our intracellular staining guide.

Summary

And that concludes our training series on flow cytometry. Hopefully, you have now developed the skills and confidence to not only design and run your flow cytometry experiments but also tackle them when things go awry.

If you feel ready, try taking our flow cytometry quiz to test your new knowledge.

Also, keep an eye on our main training page in case you need to brush up or get to get grips with other common applications in the lab.