Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6
| Autor: | Scott Powers, Robert F. Murphy, Charles R. Cantor |
|---|---|
| Rok vydání: | 1984 |
| Předmět: |
Endosome
medicine.medical_treatment media_common.quotation_subject Endosomes Biology Endocytosis Flow cytometry Rhodamine Mice chemistry.chemical_compound medicine Animals Insulin Fluorescein Internalization Cells Cultured media_common medicine.diagnostic_test Rhodamines Vesicle Articles Cell Biology Hydrogen-Ion Concentration Flow Cytometry Fluoresceins Kinetics Spectrometry Fluorescence Biochemistry chemistry Biophysics Lysosomes |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.98.5.1757 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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