I'm trying to evaluate the impact of the internalization of unlabelled crystalline silica sub-micrometric crystals in THP-1 macrophages, through lysotracker red assay, by means of flow cytometry. We works in RPMI+10%FBS, so the particles should be surrounded by serum proteins.
Whether the lysotracker fluorescence of cells incubated with particles is comparable to that of negative control (cells treated with the dye only), can we assess that the particles ARE NOT INTERNALIZED (or are internalized, but poorly) by cells? We have noticed that, using other particles (crystalline silica as well, but with different surface chemical properties), fluorescence dramatically increases.
Hi,
the accumulation of NPs in lysosomes (and thus their induction) is highly dependend on the mechanism of uptake. If NPs are taken up primarily by endocytosis-dependent mechanisms (highly dependent on their surface properties!), they usually end up in the lysosomes, which can then result in an increase of total lysosomes which you can observe by lysotracker intensity increase.
However, NPs also may cross the membrane without active uptake by the cells, and will then accumulate mostly in the cytoplasm, which without any associated secondary toxic effects (e.g. ROS induction, metal toxicity, ...) may not have any apparent effects on the cells, however, the particles are still there.
Personally, I would not consider the absence of lysosome induction as a clear proof that the particles are not taken up, however it indicates that the effects on the cells by the two NPs are different, possibly due to the uptake mechanism. If you want to have a clear and reliable result, you will either have to label your NPs or go for electron microscopy.
Lysotracker isn't used to measure uptake, it's only used to measure whether something uptaken is trafficked to the lysosome. The lysotracker labels all acidic vessicles.
The most you can say is thta there is no increase in acidic vesicles with your treatment.
As for how to interpret the increase in fluoresence from the other treatments... you still can only say that there is an increase in acidic vesicles... which may mean that the up-taken particles are being degraded, but... that's speculation.
With unlabeled particles, you might need an electron microscope.
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