|
|
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Journal of Cell Biology, Vol 98, 1757-1762, Copyright © 1984 by The Rockefeller University Press
ARTICLES |
RF Murphy, S Powers and CR Cantor
The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
