My purpose was to demonstrate the lysosomal permeabilization using acridine orange. I read a few papers on the LMP, most of them suggested the red fluorescence of acridine orange is an indicator for acidic vacuoles in cells, including lysosomes. The underlying philosophy was that when lysosomal membrane were damaged, the pH inside the lysosomes would increase, and the red flourescence will decline.
However, when I used chloroquine and hydroxychloroquine as positive controls, as these compounds increase lysosomal membrane permeability. Unexpectedly, Cells do have more red flourescence staining when excited with blue light. In this case I can not preceed with my samples, because I doubt about its specificity.
Is there anyone who had experience with this? Is it possible that CQ and HCQ just blocks the fusion between lysosomes and autophagosome, resulting in more autophagosomes stained in red? Or am I not using the right positive controls? Is there anything I have missed in this acridine orange labeling?
HCQ: 30ug/ml, 15hrs.
CQ: 100uM, 6hrs.
AO: 5ug/ml, 37 degree for 15min.
I used a BD Aria flow cytometry for the detection of both green (519nm) and red (615nm) flourescence.
AO shows green fluorescence when bound to DNA and red when bound to acidic regions which includes lysosomes. I prefer one of the Lysotrackers (red, blue, green) which do not bind to DNA so that, if there is a need for a co-localization fluorophore, another color is available.
AO shows green fluorescence when bound to d-DNA and red when bound to s-DNA
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