Troubleshooting Techniques for Flow Cytometry

This article describes in detail tips on how to deal with common issues that may arise during flow cytometry.


  1. No signal or weak fluorescence intensity
  2. High fluorescence intensity
  3. High background or a high percentage of positive cells
  4. Two or more cell populations observed when there should be one
  5. High side scatter background (from small particles)
  6. Low event rate
  7. High event rate

1. No Signal or Weak Fluorescence Intensity

The following issues may be present:

The Signal is Not Compensated in the Right Manner

Here it is important to assess that the positive single color control has been rightly set up on the flow cytometer. Ensure that it has been gated, and is properly compensated, in order to make sure that it is capable of capturing every event.

There is Too Little Antibody for Detection

In this case, the concentration or the amount of the antibody must be increased.

Intracellular target not accessible

In such a situation, the target protein must be checked to make sure it is inside the cell. The membrane must be properly permeabilized so that staining penetrates into the interior of the cell. To keep the proteins on the cell surface from being internalized, the entire procedure must be carried out on ice, or the temperature should be maintained at 4 oC, with all the reagents being ice-cold, so that all the reactions come to a stop.

If the surface antigens are to be kept from being modulated or internalized, to keep the intensity of the fluorescence intact, sodium azide may be added. If cell types need to be stained, trypsin should be substituted with gentler techniques of detachment, as it can produce internalization of the proteins on the surface of the cell, especially in cell surface molecules.

Intracellular Staining – Fluorochrome Conjugate is Too Large

Large molecular weight fluorochromes can reduce antibody motility and possibly its entry into the cell. Therefore fluorochromes for intracellular staining experiments should have low molecular weight.

Non-alignment of Lasers

During flow cytometry experiments, the lasers on the instrument must be checked for correct alignment by running flow check beads. Based on the results, the alignment may be adjusted if required. If laser alignment fails or drift is observed then the device may need to be serviced.

Target Protein Not Present/Expressed at Low Level

In such a situation, it must be verified that all the tissues or cell types used expresses the target protein at a concentration that is above detectable limits.

The Target Protein is Soluble or Secreted

If the target protein is one which dissolves or is released from the cell by secretion, the detection will be unsuccessful. The target protein must be bound to the membrane or be present in the cytoplasm for its detection by flow cytometry. Using a golgi-block step during the procedure, such as the use of Brefeldin A, could enhance the signal and make intracellular staining more efficient.

Offset Too High/Gain Too Low

Use the positive control to set up the flow cytometer correctly again, 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).

Fading of Fluorochrome Fluorescence

In this case, the antibody should be checked as it may have been exposed to the light, or may have been stored for too long. A fresh antibody will be needed.

The Primary Antibody and the Secondary Antibody are Not Compatible

Use the secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).

2. High Fluorescence Intensity

Antibody Concentration Too High

Too high of an antibody concentration will result in high levels of non-specific binding, or else excessive fluorescence intensities. To counter this, the amount of antibody added to each of the samples should be reduced.

Excess Antibody Trapped

This can be a particular problem in intracellular staining where large fluorochrome molecules on the antibody can be trapped. Ensure adequate washing steps and include tween or triton in wash buffers.

Poor Blocking

To deal with this problem, a 1 to 3% blocking agent must be added along with the antibody, and in addition to this, a blocking step must be included.

3. High Background or High Percentage of Positive Cells

Gain Set Too High or Offset Too Low

If this is the case, the flow cytometer should be set up properly again using the positive control. The offset is helpful in reducing background due to small particles and the gain can be reduced if the signal is to be decreased.

Excessive Antibody

When this occurs, the antibody concentration must be reduced. To make sure that excessive antibody is washed out, the wash buffers must contain added detergent.

4. Two or More Cell Populations Observed when There Should be Just One

More than One Cell Population Present Expressing Target Protein

In such a situation, the separation of the cells may be inadequate, so the expected level of target protein expression must be checked for each of the cell types in the sample and the cells must be well separated.

Cell Doublets are Present

When cell doublets are seen, they appear as a second population of cells with double the intensity of fluorescence shown on the plot. To resolve this, the cells should be mixed gently and then staining should be applied. Repeated mixing is required before loading them to run on the cytometer with a pipette. Another method is by sieving or filtering the cells so that any clumps are removed, using 30 micron nylon mesh.

5. The Background has High Side Scatter (from Small Particles)

Cells Lysed

To make sure that sample cells do not lyse, fresh samples and proper sample preparation is essential. High centrifugation speeds and violent vortex application to the cells must be avoided.

Bacterial Contamination

All bacteria must be excluded from the sample to avoid low-level autofluorescence from bacteria. This leads to a high event rate as well.

6. Low Event Rate

Low Number of Cells per Milliliter

To resolve this, the number of cells run should be 1x106 cells per milliliter, and the cells must be gently but thoroughly mixed.

Clumping of Cells Creating a Tubing Blockage

To make sure the cell suspension is homologous for single cells, several pipettings must be performed gently before staining is applied. A final mixing is essential before running the cytometer. If the problem is severe, the sieving or filtering of cells using a 30 micron nylon mesh will remove the clumps.

7. High Event Rate

High Number of Cells

If there are too many cells, a dilution must be applied to bring down the concentration to between 1x105 and 1x106 cells per milliliter.

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Last updated: Jun 6, 2019 at 11:54 AM


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