Journal
PLOS ONE
Volume 10, Issue 4, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0119532
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Funding
- Oxford Biomedical Research Centre - United Kingdom National Institutes for Health Research [A93081]
- ChemoCentryx, Inc., Mountain View, California, United States of America
- British Heart Foundation [RG/10/15/28578] Funding Source: researchfish
- Medical Research Council [G0200231] Funding Source: researchfish
- MRC [G0200231] Funding Source: UKRI
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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 G alpha i 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.
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