Under the excitation of the laser,some molecules and structure of cell can also produced the fluorescence,but these signal are weaker than fluorescein,and have no correlation with specific antigen molecules,so they called non-special fluorescence.

Generally speaking,the signal strength of non special fluorescence depends on the cell size,the biger,the stronger,For cance cell,it is always stonger than immune cell .

In fact,the detected data of FACS is from both special and non-special fluorescence. So it is important and necessary to set up negative control to determine the proportion of autofluorescence .

Dear Ramos,

I agree with Chen about using and setting by unlabeled cells. Some cells such as fibroblasts, alveolar macrophages or cancer cells have an autofluorescence. Some author use a kind of autofluorescence reducing agents also. You can find online.

Good luck

If you are working with a new cell line and you are unsure of their autofluorescent capacity, I would run an experiment with unstained cells under different conditions (example, normal vs stress). Run each condition unstained and with a single stain in order to see how the cells run via FACS.

Although, there are many macro-molecules within the cell line that may have some auto-fluorescence; however it is not going to affect your results once you use unstained cells as control. Furthermore, to precisely find the auto-fluorescence and non-specific binding of your antibody you should use 2 types of controls: unstained, and Isotype (IgG) conjugated with your flurophore.