Rapid measurements of intracellular calcium using a fluorescence plate reader
Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive dat...
| Veröffentlicht in: | BioTechniques. - 1991. - 26(1999), 2 vom: 01. Feb., Seite 318-22, 324-6 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1999
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, U.S. Gov't, P.H.S. Chelating Agents Fluorescent Dyes Receptor, Muscarinic M1 Receptors, Adrenergic, beta Receptors, Muscarinic Uricosuric Agents Egtazic Acid 526U7A2651 mehr... |
| Zusammenfassung: | Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells |
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| Beschreibung: | Date Completed 21.04.1999 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |