I've been trying to determine CD56, CD16, and CD49a expression on NK cells via flow cytometry. We're using a Cytek Aurora equipment and haven't been able to determine anything because the cell autofluorescence is incredibly high (10^4 or 10^5). Has any of you encountered this problem? Do you have any suggestions on how to fix it? The cells seem to be alright when I checked them on the microscope before marking, so I don't think it is because of cell death.
It is always recommended to check the condition of the cells so that they are not overgrown or stressed as this can lead to autofluorescence.
Another approach is to use a "dead cell stain" such as DAPI or PI to exclude dead cells, which can also contribute to autofluorescence.
You can also pre-treat the cells with a fixative chemical such as ethanol or formaldehyde that can quench the autofluorescence and fix the protein of interest in the cells.
Lastly, you should check the settings of the instrument and make sure that the laser settings are set correctly and that the settings are optimal for your samples and reagents.
Hello,
Do you see this strong autofluorescence under the microscope?
As suggested above, I would check the instrument settings such as laser strength.
Check also your antibody fluorescence on compensation beads. With adjusted laser strength, their signal should be stronger than the autofluorescence.
You might wish to check the availability of magnetic beads containing the same antibodies and use them since auto fluorescence should not be due to the same cell surface proteins as those you are seeking to measure with flow cytometry.
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