How to distinguish phagocytosed particles from attached ones - (Jul/28/2006 )

Hi,
I am studying phagocytosis of fluorescent particles by macrophage derived cell line, and analyzing the data using flow cytometry. But the process is not able to distinguish between the cells with the adherent particles and internalized particles. I need to remove the partilces adhered to the cell surface completely or to quantitate the percent of particles adhrered or internalized. I shall be grateful if anyone can guide me in this respect.
Thanks.
Yogesh

First, have you tried your best to wash/compete off /or even trypsin treatments to remove the attached beads?

You may use an antibody with a different label to bind exposed beads. If you chose right label combination, you may be able to quantify the accessible beads using Fluorescence Resonance Energy Transfer principle. You need to discuss this with the companies see it is possible for this application.

You can always adopt this system with fluorescence microscopy.

in my old job, we had the same problem... until we found an article that uses trypan blue to quench the FITC-signal towards FL-3... we used FITC-labeled bacteria that should be phagocytosed by cells... and indeed you can't distinguish between internalised and attached FITC-labeled bacteria...
now we just did the experiment the same way we always did with the FITC-labeled bacteria and measure it in a flow cytometer, and than add trypan blue and measure it again now you can distinguish between the bound and phagocytosed bacteria...
to be short: trypan blue quenches the FITC signal from FL-1 to FL-3... and since trypan blue will not enter a living cell, you still get your FITC-signal for the phagocytosed bacteria and the quenched signal for the attached bacteria...
i'm sure this will also work on other particles as log as it's FITC-labeled....

Thanks a lot.