| Low fluorescence signal | - Is the signal correctly compensated for? Ensure the positive single color control is set correctly on the instrument, gated, and compensated for to capture all the events.
- Is the amount of antibody sufficient for detection? Try increasing the amount and/or concentration of antibodies and retest.
- If not present at all, ensure the tissue and/or cell type expresses the target protein. Ensure the tissue and/or cell type is present in a high enough concentration (around 1x10e6 cells).
- If the target molecule is scarce, a bright fluorophore may be needed.
- If the antigen is intracellular, cells require fixing and permeabilizing for antibodies to access epitopes.
- Make sure that the fluorophores are matched with the flow cytometer lasers and detectors, and are protected from light to prevent photobleaching.
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| High background noise present | - Is the gain set too high, or the offset too low? Adjusting the positive control offset will reduce the background, and reducing the gain will decrease the signal.
- High background can also result from inadequate blocking, washing, or antibody concentration.
- High background may be due to autofluorescence, to ensure cells are not over-fixed to minimize the presence of dead cells. Use red fluorophores for detecting highly autofluorescent cell types, like neutrophils. Keep samples on ice, avoid freeze-thawing, and include a viability dye to prevent/minimize dead cells.
- Are you detecting non-specific cell types? Use well-validated antibodies where possible, and if necessary, introduce an Fc blocking step into the experiment.
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| Fluorescence intensity is too high | - Is the antibody concentration too high, leading to non-specific binding? Reduce the antibody concentration, and retest.
- Are instrument settings correct? Try decreasing the laser power, reducing the gain, or optimizing the photomultiplier tube (PMT) voltages.
- Modifications to the blocking step may be needed, e.g., switch to a different blocking solution, or increase the blocking time.
- Modification to washing steps may be needed, should excess antibody be trapped within the sample. Try increasing washing steps, or include a low concentration of tween, triton, or similar detergent into the buffer.
- Ensure that extremely abundant antigens are paired with dimmer fluorophores.
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| Intracellular target does not appear during analysis | - Is the target protein intracellular? For internal staining, ensure adequate permeabilization. Ensure all reagents and steps are performed on ice.
- Gentler detachment methods may be required, and may require the addition of sodium azide which will prevent the internalization of surface antigens. When staining cell lines, trypsin may induce internalization of cell surface proteins.
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| Fluorochrome conjugate is too large | These fluorochromes should have a low molecular weight, as larger ones can reduce antibody motility and prevent entry into the cell. |
