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Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

Wendt ER, Ferry H, Greaves DR, Keshav S - PLoS ONE (2015)

Bottom Line: Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction.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.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.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Experimental Medicine Division, Translational Gastroenterology Unit, University of Oxford, Oxford, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Analysis of calcium flux by discrete populations of primary cells.Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Calcium flux in response to CCL20 (125 ng/ml) or ionomycin (5 μg/ml) was monitored by flow cytometry. (A) Gating strategy to exclude non-viable cells, select singlets, and distinguish CCR6+ from CCR6- cells. (B) Calcium flux depicted as the mean value of the Fura Red Ratio over time, in response to CCL20 or ionomycin. (C) Calcium response depicted as the percent of cells responding to stimuli greater than the average background signal. One representative experiment of n = 5 shown, performed with a minimum of two technical replicates.
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pone.0119532.g003: Analysis of calcium flux by discrete populations of primary cells.Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Calcium flux in response to CCL20 (125 ng/ml) or ionomycin (5 μg/ml) was monitored by flow cytometry. (A) Gating strategy to exclude non-viable cells, select singlets, and distinguish CCR6+ from CCR6- cells. (B) Calcium flux depicted as the mean value of the Fura Red Ratio over time, in response to CCL20 or ionomycin. (C) Calcium response depicted as the percent of cells responding to stimuli greater than the average background signal. One representative experiment of n = 5 shown, performed with a minimum of two technical replicates.

Mentions: To demonstrate the utility of calcium flux monitored by flow cytometry, we used primary human T cells stained for CCR6, and monitored calcium flux in response to CCL20. CCL20 is the single ligand to CCR6, and a subset of peripheral blood T cells express surface CCR6 [19]. Fig 3A illustrates a gating strategy to exclude non-viable cells and cell doublets, and to selectively gate CCR6+ cells.


Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

Wendt ER, Ferry H, Greaves DR, Keshav S - PLoS ONE (2015)

Analysis of calcium flux by discrete populations of primary cells.Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Calcium flux in response to CCL20 (125 ng/ml) or ionomycin (5 μg/ml) was monitored by flow cytometry. (A) Gating strategy to exclude non-viable cells, select singlets, and distinguish CCR6+ from CCR6- cells. (B) Calcium flux depicted as the mean value of the Fura Red Ratio over time, in response to CCL20 or ionomycin. (C) Calcium response depicted as the percent of cells responding to stimuli greater than the average background signal. One representative experiment of n = 5 shown, performed with a minimum of two technical replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383592&req=5

pone.0119532.g003: Analysis of calcium flux by discrete populations of primary cells.Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Calcium flux in response to CCL20 (125 ng/ml) or ionomycin (5 μg/ml) was monitored by flow cytometry. (A) Gating strategy to exclude non-viable cells, select singlets, and distinguish CCR6+ from CCR6- cells. (B) Calcium flux depicted as the mean value of the Fura Red Ratio over time, in response to CCL20 or ionomycin. (C) Calcium response depicted as the percent of cells responding to stimuli greater than the average background signal. One representative experiment of n = 5 shown, performed with a minimum of two technical replicates.
Mentions: To demonstrate the utility of calcium flux monitored by flow cytometry, we used primary human T cells stained for CCR6, and monitored calcium flux in response to CCL20. CCL20 is the single ligand to CCR6, and a subset of peripheral blood T cells express surface CCR6 [19]. Fig 3A illustrates a gating strategy to exclude non-viable cells and cell doublets, and to selectively gate CCR6+ cells.

Bottom Line: Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction.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.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.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Experimental Medicine Division, Translational Gastroenterology Unit, University of Oxford, Oxford, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus