Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6

Murphy, R. F., Powers, S., Cantor, C. R. (May 1984) Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. Journal of Cell Biology, 98 (5). pp. 1757-62. ISSN 0021-9525

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URL: http://www.ncbi.nlm.nih.gov/pubmed/6144684

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Animals Cells, Cultured *Endocytosis Endosomes/*physiology Flow Cytometry Fluorescein Fluoresceins Hydrogen-Ion Concentration Insulin/metabolism Kinetics Lysosomes/physiology Mice Rhodamines Spectrometry, Fluorescence
Subjects: Investigative techniques and equipment > flow cytometry
CSHL Authors:
Communities: CSHL labs > Powers lab
Depositing User: Matt Covey
Date: May 1984
Date Deposited: 26 Feb 2014 16:50
Last Modified: 26 Feb 2014 16:50
PMCID: PMC2113196
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29510

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