A subset of my HL60 cells has red autofluorescence in the far red region (FL-3 > 670 nm). I first noticed this some months ago when I was checking an antibody with a PE-Cy5 tag by flow cytometry. The red autofluorescence could be verified by fluorescence microscope (see picture). At first I thought it was contamination and reran with new samples. I changed to new reagents and media. I also checked a different stock and ordered new cells from ATCC, but the red cells are still there. We have never transfected these cells and we have not worked with other cells in this period. I have also tried to culture in phenol red-free media to see if they absorb the dye, but after one week the cells still had the red signal and it was not any weaker.
I am wondering if this could be something that the cells produce? Did any of you have similar observations? Can you please help me explain what this might be?
Dear Anja!
You can see in this practical guide, please:
https://www.aatbio.com/resources/assaywise/2017-6-2/a-practical-guide-for-use-of-pe-and-apc-in-flow-cytometry
Have you only used one PE-Cy5 dye?
If there were two dyes, then it is quite possible that a compensation is required.
Usually, PE-Cy5-labeled secondary antibodies are used.
If these were PE-Cy5-labeled secondary antibodies, then which protein were the primary antibodies to?
No, it is not a problem with the PE-Cy5 antibody or the flow cytometry analysis, but the unstained cells are fluorescent in the same channel.
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