assay protocol

Flow Cytometry Troubleshooting

No staining 1.Checkifantibodies have been stored at -20°C before use orexceeded their dateof expiration.
2.Checkif appropriate primary or secondary antibodies havebeen added.
3. Check if antibody is conjugated to a fluorochrome. If not,ensure that appropriate fluorochrome-conjugated secondary isbeing used.
4.Ensure that correct secondary antibody is being used including right species and Ig type.
5.If the fluorochromeused is Phycoerythrin or Allophycocyaninbased,ensure that the product has not been frozen.
6.Is the target antigen existed in your sample? Check literature forantigen expression and incorporate a positive control of knownantigen expression alongside samples.
7.Does antibody recognize antigen in test species? Check thatantibody cross-reacts with species being used. Not all antibodieswill cross-react across species.
8.Ensure that correct laser is being used to excite fluorochrome, andthat correct channel is being used to analyze emissions.
PE antibody does not stainbut same FITC antibody givesgood results. 1.PE conjugate may have been frozen. If so, purchase another vialof antibody.
2.Paraformaldehyde (PFA) may be a problem. Breakdown of PFAmay release methanol, which will affect staining. Make up freshparaformaldehyde. Cells can be analyzed immediately withoutfixing.
Non-specific staining 1.Non-specific staining may be due to autofluorescence. Solution:check levels of autofluorescenceby including a tube of cells only(i.e. without any antibody) into your panel.
2.Certain cells express low affinity Fc receptors CD16/CD32, whichbind whole antibodies via Fc region. For mouse cells, diluteantibody in SeroBlockFcR.
3.Non-specific staining may be due to the secondary antibody.Select a secondary antibody that willnot cross-react with targettissue.
4.Ensure that washing steps have been sufficient.
Weak staining 1.Weak staining may be due to overdilution of antibodies. Ensurethat antibodies are used at the correct concentration by titratingantibodies before use.
2.Weak staining in indirect staining systems may be due toprozoningeffect, where highly concentrated antibodies may giveweak results. Titrateantibodies carefully.
3.Weak staining may be due to an excess cell number. Adjust cellpopulation to recommended density.
4.Weak staining may be due to the antigen expression. Checkliterature for expected levels of expression.
5.If antigen expression is weak, select an antibody that isconjugated to a brighter fluorochrome.
6.Weak staining may be seen if using a cross-reacting antibodyrather than one specific for the target species.
7.Incubation time and temperature with either primary orsecondary antibody should be optimized.
Unusual scatter profiles 1.Ensure that cells are used as fresh as possible. Profile may beshowing dead cells and debris.
2.Activation methods may affect scatter characteristics of cells.
3.If you are using lysing solution, ensure that this is fresh and hasbeen made up correctly.
Unexpected staining 1.Some reagents may affect certain antigens and, therefore, mayneed reviewing e.g. EDTA will affect some platelet markers.
2.Lysing solutions may affect certain antigens. Select a method thatdoes not interfere with antigen detection.
3.Some antigens are expressed intracellularly and, therefore, cellpermeabilizationmethods may be required. Check manufacturer'sdatasheet for correct permeabilizationreagent.