There's no single reason why macrophages or other myeloid-derived cells autofluoresce, but some of the varied reasons may include the presence and concentration of NAD(P)H, flavins, porphyrin, or lipofuscin.

Personally, I've noticed that it gets worse when macrophages are primed/activated with LPS.

Here is a paper that discusses more: http://www.jleukbio.org/content/88/3/597.full

In the JLB paper that Jordan has mentioned we discussed potential autofluorescence sources based on the literature but we never actually went in and systematically investigated this.

One really important thing that I don't think was mentioned in the paper is that tissue resident macrophages are long lived and continue to get vastly more fluorescent with age. This really seems to fit with the idea that they are full of tissue breakdown products - probably lipofuscin etc.

At my previous position (Temple Lung Center) we noticed that the lung alveolar macrophages obtained from human patients exhibited considerably higher autofluorescence (1-2 decades) compared with alveolar macs (AMs) from the lungs of laboratory rats and mice. Flow cytometry was doable with the rodent AMs, but very challenging/not possible with the human cells. We speculated that it was likely due to accumulation of inhaled particulates over many years (the cells are long-lived as mentioned above) breathing polluted air, while laboratory animals have much shorter life spans during which they breathe filtered air. Not surprisingly, the AMs from smokers are even more autofluorescent (more inhaled particulates). So, while most macrophages aren't directly exposed to the environment like AMs, they are all sort of cellular garbage cans that will tend to accumulate foreign materials in the body over time - many of which may be highly autofluorescent.

It is important to note that macrophages have more complexity than monocytes, even in terms of mitochondrial content.

However, all cells exhibit autofluorescence when analized by flow cytometry. Even so, autofluorescence does not interfere with analyses, since you must always compare basal fluorescence with fluorescence of antibodies or fluorophores used in you assays.

Kind regards,

Raphael

The autofluorescence of the cells is usually due to the presence of coenzymes such as NAD, Flavines or other chromoproteins. In particular in macrophages the number of these molecules can be higher than in other cells because of the presence of a remarkable number of mitochondria. It is important to compare the basal autofluorescence of the macrophages with the one of the same cells treated with a fluorescent antibody

Each time you decide to investigate fluorescence from monocytes-macrophages, you have to double check the potential endogenous fluorophores that you might excite (NADH , NADPH, FADH, FADPH...). Monocytes macrophages, polymorphonuclear neutrophils, mast cells, lymphocytes are auto fluorescent in specific and different wavelength. I agree with Angela saying that we always would have to normalize fluorescent intensity coming from a fluorescent labeling system, to the basal auto fluorescent level. And I also agree with Jordan: change in energetic metabolism, or inflammatory conditions can definitely impact this basal fluorescence. See the enclosed article.