I am differentiating mouse bone marrow macrophages in vitro. However, I found that the differentiated macrophages have higher background fluorescence (the unstained sample) in all channels, which includes both BV421 and AF-647. I also test this in single-stained cells and got similar results (therefore, I believe is not because of failed compensation).

I have these specific questions:

  1. Is it common to see cells having different background fluorescence?
  2. Are there any tools or algorithms to be used to correct such differences?
  3. For publication/ presentation purposes, is it common to omit the plot of unstained control? I would like to know what is the convention.

I hope this is not caused by my experiment's failure. I found this thread, but the hyperlink does not work. Therefore, it would be great if you can also comment on what are the potential causes of this observation if you found that relevant.


  • $\begingroup$ Far from an expert here but I believe that there are many tools that can help you correct background fluorescence, here is one such. $\endgroup$ Commented Feb 2, 2023 at 19:18


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