Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.
Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantage...
| Main Authors: | , , , |
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| Format: | Journal article |
| Language: | English |
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Public Library of Science
2015
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| _version_ | 1797076019160547328 |
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| author | Wendt, E Ferry, H Greaves, D Keshav, S |
| author_facet | Wendt, E Ferry, H Greaves, D Keshav, S |
| author_sort | Wendt, E |
| collection | OXFORD |
| description | Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1) using a single calcium dye provides an additional channel for surface marker characterization, 2) allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3) can measure total calcium flux and additionally, the proportion of responding cells, 4) can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX), on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells. |
| first_indexed | 2024-03-06T23:58:15Z |
| format | Journal article |
| id | oxford-uuid:74ff6f49-6881-45b6-9c74-6af63005c3ab |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T23:58:15Z |
| publishDate | 2015 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | oxford-uuid:74ff6f49-6881-45b6-9c74-6af63005c3ab2022-03-26T20:06:38ZRatiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:74ff6f49-6881-45b6-9c74-6af63005c3abEnglishSymplectic Elements at OxfordPublic Library of Science2015Wendt, EFerry, HGreaves, DKeshav, SCalcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1) using a single calcium dye provides an additional channel for surface marker characterization, 2) allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3) can measure total calcium flux and additionally, the proportion of responding cells, 4) can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX), on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells. |
| spellingShingle | Wendt, E Ferry, H Greaves, D Keshav, S Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title | Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title_full | Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title_fullStr | Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title_full_unstemmed | Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title_short | Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets. |
| title_sort | ratiometric analysis of fura red by flow cytometry a technique for monitoring intracellular calcium flux in primary cell subsets |
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