We are using pHrodo Red Avidin to follow (by Flow Cytometry) the endocytosis of biotinylated antibodies reacting with mouse dendritic cell receptors. Fluorescence after incubation at 37°C does not differ significantly from that of cells incubated at 4°C. We do not have any significant shifts when we use biotinylated zymosan (or biotinylated E. coli) as positive controls. We are using a FACScalibur, FL2 Channel - Blue Laser (488nm) and 564-601nm Emission Filter. Has anyone tried pHrodo Red Avidin, or have any suggestions?
One problem with your particular setup is that you are using DC (immature or mature?) that may not have that acidic endosomes so pHrodo may not give much of a difference at these different pHs. Another particular problem, which will also depend the sizes of these immune complexes, is if the IgG-avidin can be recycled by FcRn, thus simply be shuttled back to the surface fairly quickly. The IgG-Avidin complexes are likely to be bound by FcgR on the surface at both 4°C and 37°C (you may get rid of this by acid elution), and these , again depending on the sizes of the immune complexes, may trigger phagocytosis or endocytosis, leading to phago-lysosomal destruction, or as I mentioned earlier, to FcRn-dependent recycling. Hope this helps.
Hi Gestur, thank you very much for your comments. We are working with BMDC mostly with an immature phenotype. I was not aware of the fact that they may not have acidic endosomes, but this is an interesting point. Could you suggest a paper to gain insight into this matter? Regarding your comments on nFcR, it may not apply to this case because we are using recombinant nanobodies that lack the Fc region. Sorry for missing this information in original question.
HI Gualberto, just to confirm.... I work on monocytes derived dendritic cell, focusing on lysosomal activity. I tryed Ph rodo but doesn't work as the ph of lysosomal compartment is not enough acid to give a nice shift. If you want to have confirmation you should try to compare your DCs with macrophages, they have a real acid ph. You can also try to use fitc-dextran, many different size available and the experiment are very easy. One of the best author for lysosome in Dendritic cell is Colin Watts.
Hope it was helpful
Thanks Dario, it is nice to know that from your own experience it does not work with DCs. We can try our Abs in macrophages with Phrodo and then go back to DCs with fitc.
Hi Gualberto and Leone,
I would then rather go for Alexa488 or a Dy analogues as FITC is quite sensitive to slight acid environment (DC´s do acidify but indeed much less than monocytes explaining your pHrodo results).
Good luck!
Gestur
Just a few thoughts:
1) pHrodo red is weakly excited by the 488nm laser. You will gain considerably sensitivity y exciting with a yellow laser (561nm). Alternatively, use pHrodo green.
2) As others mentions, DCs do not acidify as much as macrophages. They counteract the acidification of their phagosomes by using NADPH Oxidase to pump in reactive oxygen species which help to alkalinize the phagosome (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597138/) Perhaps you could use something like Fc.Oxyburst to monitor endocytosis.
3) Use Trypan Blue to quench extracellular fluorescence (bound but not engulfed). Alternatively, increase the pH of your FACS buffer to limit fluorescence from extracellular fluorophore. While pHrodo is weakly fluorescent at neutral pH, it is still fluorescent.
4) Make sure there is no trace amount of biotin in any of the solutions that the avidin has contact with.
Thanks Evan. We are holding this project for a while, but your comments will be useful when we resume.
Hi Gualberto,
Reading from your post, I realized you have worked with biotinylated zymosan. Could you please share your protocol for generating biotinylated zymosan.
Thank you!
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