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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

Identifying channels available in combination with Fura Red calcium dye.Expanded human T cells were loaded with 1 μM Fura Red, AM for 30 minutes. Calcium flux in response to ionomycin (5 μg/ml added at 25 seconds) was recorded on all available channels using an LSRII SORP flow cytometer. Cells were selectively gated on lymphocytes and singlets. The names assigned to the different detectors represent fluorochromes that are commonly detected in that channel. (A) Channels used to perform ratiometric analysis of Fura Red dye. (B) Representative examples of channels that display a shift in MFI following calcium mobilization. Channels such as these cannot be used for surface protein characterization in combination with Fura Red. (C) Channels with no change in MFI following calcium mobilization. Such channels are available for surface marker and cell viability analysis in combination with Fura Red dye. On the tested LSRII SORP, four available channels were identified, and fluorochromes commonly detected in these channels include: AlexFluor488, APC, APC-Cy7, and AlexaFluor700. One representative experiment of n = 2 shown.
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pone.0119532.g002: Identifying channels available in combination with Fura Red calcium dye.Expanded human T cells were loaded with 1 μM Fura Red, AM for 30 minutes. Calcium flux in response to ionomycin (5 μg/ml added at 25 seconds) was recorded on all available channels using an LSRII SORP flow cytometer. Cells were selectively gated on lymphocytes and singlets. The names assigned to the different detectors represent fluorochromes that are commonly detected in that channel. (A) Channels used to perform ratiometric analysis of Fura Red dye. (B) Representative examples of channels that display a shift in MFI following calcium mobilization. Channels such as these cannot be used for surface protein characterization in combination with Fura Red. (C) Channels with no change in MFI following calcium mobilization. Such channels are available for surface marker and cell viability analysis in combination with Fura Red dye. On the tested LSRII SORP, four available channels were identified, and fluorochromes commonly detected in these channels include: AlexFluor488, APC, APC-Cy7, and AlexaFluor700. One representative experiment of n = 2 shown.

Mentions: Primary human T cells were loaded with Fura Red, AM, stimulated with ionomycin, and changes to mean fluorescence intensity (MFI) over time was measured on all available channels on an LSRII SORP. Ratiometric detection of Fura Red was monitored off the Violet and Green lasers, as advised by the manufacturer (Fig 2A). Fig 2B provides representative traces for channels that display shifts in MFI following calcium mobilization. These channels are therefore not suitable for surface antigen characterization. Channels that do not display a shift in MFI following calcium flux can be used for staining with a viability dye or for detecting surface antigens (Fig 2C). On the LSRII SORP, four available channels were identified, and fluorochromes commonly detected by these channels include: AlexaFluor488, APC, APC-Cy, and AlexaFluor700.


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)

Identifying channels available in combination with Fura Red calcium dye.Expanded human T cells were loaded with 1 μM Fura Red, AM for 30 minutes. Calcium flux in response to ionomycin (5 μg/ml added at 25 seconds) was recorded on all available channels using an LSRII SORP flow cytometer. Cells were selectively gated on lymphocytes and singlets. The names assigned to the different detectors represent fluorochromes that are commonly detected in that channel. (A) Channels used to perform ratiometric analysis of Fura Red dye. (B) Representative examples of channels that display a shift in MFI following calcium mobilization. Channels such as these cannot be used for surface protein characterization in combination with Fura Red. (C) Channels with no change in MFI following calcium mobilization. Such channels are available for surface marker and cell viability analysis in combination with Fura Red dye. On the tested LSRII SORP, four available channels were identified, and fluorochromes commonly detected in these channels include: AlexFluor488, APC, APC-Cy7, and AlexaFluor700. One representative experiment of n = 2 shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119532.g002: Identifying channels available in combination with Fura Red calcium dye.Expanded human T cells were loaded with 1 μM Fura Red, AM for 30 minutes. Calcium flux in response to ionomycin (5 μg/ml added at 25 seconds) was recorded on all available channels using an LSRII SORP flow cytometer. Cells were selectively gated on lymphocytes and singlets. The names assigned to the different detectors represent fluorochromes that are commonly detected in that channel. (A) Channels used to perform ratiometric analysis of Fura Red dye. (B) Representative examples of channels that display a shift in MFI following calcium mobilization. Channels such as these cannot be used for surface protein characterization in combination with Fura Red. (C) Channels with no change in MFI following calcium mobilization. Such channels are available for surface marker and cell viability analysis in combination with Fura Red dye. On the tested LSRII SORP, four available channels were identified, and fluorochromes commonly detected in these channels include: AlexFluor488, APC, APC-Cy7, and AlexaFluor700. One representative experiment of n = 2 shown.
Mentions: Primary human T cells were loaded with Fura Red, AM, stimulated with ionomycin, and changes to mean fluorescence intensity (MFI) over time was measured on all available channels on an LSRII SORP. Ratiometric detection of Fura Red was monitored off the Violet and Green lasers, as advised by the manufacturer (Fig 2A). Fig 2B provides representative traces for channels that display shifts in MFI following calcium mobilization. These channels are therefore not suitable for surface antigen characterization. Channels that do not display a shift in MFI following calcium flux can be used for staining with a viability dye or for detecting surface antigens (Fig 2C). On the LSRII SORP, four available channels were identified, and fluorochromes commonly detected by these channels include: AlexaFluor488, APC, APC-Cy, and AlexaFluor700.

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