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FCM Troubleshooting

1. No staining

Ensure that all the primary and secondary have been correctly stored and have activity;

Set a positive control of known antigen expression to expel other unexpected reasong that may result in false-negative;

Don't freeze your materials if the fluorescent dye is phycoerythrin or allophycocyanin-based.

2. Weak staining

Maybe the concentration of antibody is too low. Ensure you have used the suitable concentration;

Maybe the number of cells is too high. Adjust cell density to recommended one;

Maybe the incubated time and temperature with antibody is not suitable, which should be optimizaed;

Maybe the antigen expression is weak. Select an antibody that is conjugated to a brighter fluorochrome, such as PE and PerCP.

3. Nonspecific staining

Maybe the concentration of antibody is too low, which should be optimized carefully;

In indirect IF staining, select a secondary antibody that do not have cross-react with your material;

Make sure that you have blocking nonspecific binding sites and have adequate washing steps.

4. Unexpected staining

Make sure that your reagents used don't affect the activity or structure of your antigens;

If your tested antigens are expressed intracellularly, some cell permeabilization methods should be used.