Why does my unstimulated sample looks stimulated after FACS acqusition?

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Nicolò M. Villa

Dear Nam Tae Kim ,

There are several possible reasons why your unstimulated sample looks stimulated after FACS acquisition:

Instrument issues: It is possible that there may be an issue with the FACS instrument, such as fluorescence spillover or laser alignment, which could cause the unstimulated sample to appear stimulated.

Autofluorescence: Some cell types or sample components can exhibit autofluorescence, which could lead to false-positive signals in the unstimulated sample.

Non-specific binding: Antibodies used for surface staining may bind non-specifically to cells, leading to false-positive signals.

Contamination: Cross-contamination between samples can occur during FACS acquisition, which can result in false-positive signals in the unstimulated sample.

It is important to carefully examine your FACS acquisition settings and gating strategy to ensure that the signals you are seeing are not artifacts. Additionally, including appropriate controls, such as fluorescence-minus-one (FMO) controls, can help to distinguish true-positive signals from background noise.

Nam Tae Kim

Thank you so much for these reasons I could rule out! I am doing this the past months and there's nothing wrong with my results. I changed some of the fluorochromes I used because we ran out of them and after going back to my original panel, this happened. Is there a possibility that changing my panel affects my results?