No or weak signal in Flow cytometry

Signal not correctly compensated

Within a flow cytometer excitation and emission wavelengths are selected by bandpass filters. To correct for spectral overlap, fluorescence should be compensated so that what is detected comes from the flurorchrome being measured.

• Check positive single color control is set up correctly on flow cytometer, gated and compensated correctly to capture all the events.

• Increase amount/concentration of antibody.

• Check if target protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent internalisation of cell surface proteins, everything must be done on ice or at 4°C, with ice cold reagents, to stop all reactions.

• Adding sodium azide will prevent the modulation and internalization of surface antigens which can produce a loss of fluorescence intensity.

• For staining of cell lines, trypsin can often induce internalisation of cell surface proteins, particularly cell surface molecules, 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. Fluorochromes with large molecular weights can reduce antibody motility and its ability to enter the cell.

• Test using a fluorochrome with a lower molecular weight.

Lasers not aligned.

Within benchtop flow cytometers no operator adjustment is needed, however steam-in-air flow cytometers may need to have lasers adjusted to ensure it focuses on your sample core. This requires laser safety training and in most cases should be left to service engineers but if your alignement issues are consistent it may be best to have the machine serviced.

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

• Ensure tissue/cell type expresses target protein and that it is present in a high enough amount to detect.

Soluble/secreted target protein

The target protein needs to be soluble and secreted from the cell (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.

• 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).

Fluorochrome fluorescence has faded.

Antibody may have been kept for too long or left out in the light.

• Fresh antibody will be required.

The primary antibody and the secondary antibody are not compatible

Secondary antibodies should be carefully matched to your primary antibody across different characteristics. If you're struggling to find the right secondary antibody, read our guide on selecting a secondary.

• Use secondary antibody that was raised against the species in which the primary was raised (eg if the primary is raised in rabbit, use an anti-rabbit secondary).