Troubleshooting
If something doesn’t work, check through the following guidelines to identify and resolve the problem. If you are still experiencing difficulties and have purchased your reagents from Bio-Rad, our
Technical Services Team will be happy to offer further advice.
Problem
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Course of Action
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No staining
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Confirm that all antibodies have been stored correctly according to the manufacturer’s instructions.
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Confirm that commercial antibodies have not exceeded their date of expiration.
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Make sure that appropriate primary or secondary antibodies have been added.
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Make sure that antibody is conjugated to a fluorophore. If not, confirm that an appropriate fluorophore-conjugated secondary antibody is being used.
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Confirm that secondary antibody is active. Has it been used successfully with other primary antibodies?
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Make sure a secondary antibody that will recognize your primary antibody is being used.
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If the fluorophore used is phycoerythrin or allophycocyanin based, make sure that the product has not been frozen.
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Is the target antigen present on test tissue? Check literature for antigen expression and incorporate a positive control of known antigen expression alongside test material.
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Does antibody recognize antigen in test species? Check that antibody cross-reacts with species being used. Not all antibodies will react across species.
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Confirm that correct laser is being used to excite fluorophore, and that correct channel is being used to analyze emissions.
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No staining with PE antibody but same FITC antibody gives good results.
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PE conjugate may have been frozen. If so, purchase another vial of antibody.
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Paraformaldehyde (PFA) may be a problem. Breakdown of PFA may release methanol, which will affect staining. Make up fresh PFA. Cells can be analyzed immediately without fixing.
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Non-specific staining
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May be due to autofluorescence. Solution: check levels of autofluorescence by including only a tube of cells (that is, without any antibody) in your panel.
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Certain cells express low-affinity Fc receptors CD16/CD32, which bind whole antibodies via Fc region. For mouse cells, dilute antibody in Mouse SeroBlock FcR (Bio-Rad product code BUF041A or B).
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May be due to the secondary antibody. Select a secondary antibody that will not cross-react with target tissue.
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Make sure that sufficient washing steps have been included.
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Titrate test antibody carefully. Nonspecific staining may be reduced at lower antibody concentrations.
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Weak staining
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May be due to overdilution of antibodies. Confirm that antibodies are used at the correct concentration by titrating antibodies before use.
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Weak staining in indirect staining systems may be due to prozoning effect, where highly concentrated antibodies may give weak results. Titrate antibodies carefully.
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May be due to an excessive number of cells. Adjust cell population to recommended density.
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May be due to the antigen expression. Check literature for expected levels of expression.
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If antigen expression is weak, select an antibody that is conjugated to a brighter fluorophore.
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May be seen if using a cross-reacting antibody rather than one specific for the target species.
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Optimize incubation time and temperature with either primary or secondary antibody.
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Unusual scatter profiles
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Make sure that cells as fresh as possible are used. Profile may be showing dead cells and debris.
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Activation methods may affect scatter characteristics of cells.
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If you are using lysing solution, confirm that this is fresh and has been made up correctly.
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Unexpected staining
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Some reagents may affect certain antigens and, therefore, may need reviewing. For example, EDTA will affect some platelet markers.
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Lysing solutions may affect certain antigens. Select a method that does not interfere with antigen detection.
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Some antigens are expressed intracellularly, therefore cell permeabilization methods may be required. Check manufacturer’s datasheet for correct permeabilization reagent.
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