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Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression.

Kenty JH, Melton DA - PLoS ONE (2015)

Bottom Line: Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression.Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells.Human islet cells were also assessed and showed functional α and β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.

No MeSH data available.


A rise in intracellular calcium corresponds to insulin secretion.(A) Overview of calcium imaging method for islets: intact islets were plated on a 96 well plate. Islets were stained with the Fluo-4 AM and then washed in fasting solution. Calcium influx of the population of cells in the islet or single cells was imaged following the addition of glucose. Same islets were imaged again after fixation and immunofluorescence staining with INS, GCG, and NKX6.1. (B) Representative images of WT mouse islets after stimulating with 2.5 and 15 mM glucose followed by 30 mM KCl. (C) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity for WT mouse islets shown in Fig 1B (7 islets) with corresponding S1 Movie. The calcium influx response for each mouse islet was normalized to the starting fluorescence intensity data point during the initial low glucose incubation. The normalized fluorescence intensities of 7 WT mouse islets were averaged and plotted on the y-axis with standard deviations. Islets were challenged sequentially with 2.5, 15, 2.5, 15, 2.5, and 15 mM glucose and 30 mM KCl. Fluorescence was measured at 126 time points throughout the series of glucose challenges, normalized to the starting fluorescence intensity, and averaged across all islets at each time point. The standard deviation at each time point ranged between ± 2 to 8 a.u.c (area under the curve). The x-axis represents time (in seconds). P-value was calculated from the difference between ave a.u.c. during low glucose and high glucose stimulations. Significance of calcium influx response was labeled * when the P-value was between 0.05 and 0.001 and ** when the P-value was below 0.001. (D) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity of a total of eight WT mouse islets with corresponding S2 Movie. Population measurements of dynamic normalized Fluo-4 fluorescence intensity for mouse islets is shown in purple, and the ELISA measurements of secreted mouse insulin for the same batch of islets is shown in blue. Challenges were done with 5 minutes of 2.5 mM, 25 minutes of 15 mM, and 5 minutes of 30 mM KCl.
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pone.0122044.g001: A rise in intracellular calcium corresponds to insulin secretion.(A) Overview of calcium imaging method for islets: intact islets were plated on a 96 well plate. Islets were stained with the Fluo-4 AM and then washed in fasting solution. Calcium influx of the population of cells in the islet or single cells was imaged following the addition of glucose. Same islets were imaged again after fixation and immunofluorescence staining with INS, GCG, and NKX6.1. (B) Representative images of WT mouse islets after stimulating with 2.5 and 15 mM glucose followed by 30 mM KCl. (C) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity for WT mouse islets shown in Fig 1B (7 islets) with corresponding S1 Movie. The calcium influx response for each mouse islet was normalized to the starting fluorescence intensity data point during the initial low glucose incubation. The normalized fluorescence intensities of 7 WT mouse islets were averaged and plotted on the y-axis with standard deviations. Islets were challenged sequentially with 2.5, 15, 2.5, 15, 2.5, and 15 mM glucose and 30 mM KCl. Fluorescence was measured at 126 time points throughout the series of glucose challenges, normalized to the starting fluorescence intensity, and averaged across all islets at each time point. The standard deviation at each time point ranged between ± 2 to 8 a.u.c (area under the curve). The x-axis represents time (in seconds). P-value was calculated from the difference between ave a.u.c. during low glucose and high glucose stimulations. Significance of calcium influx response was labeled * when the P-value was between 0.05 and 0.001 and ** when the P-value was below 0.001. (D) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity of a total of eight WT mouse islets with corresponding S2 Movie. Population measurements of dynamic normalized Fluo-4 fluorescence intensity for mouse islets is shown in purple, and the ELISA measurements of secreted mouse insulin for the same batch of islets is shown in blue. Challenges were done with 5 minutes of 2.5 mM, 25 minutes of 15 mM, and 5 minutes of 30 mM KCl.

Mentions: Towards the goal of assessing the function of healthy and diseased islets, a method was developed to image calcium flux of whole islets (population analysis) and individual islet cells (single cell analysis) in intact islets followed by fixation and immunostaining to examine marker expression profiles of calcium-imaged cells (Fig 1A). Purified islets were loaded with the indicator, Fluo-4 AM, which fluoresces upon binding calcium. Islets were imaged while being stimulated with a series of sequential glucose challenges: low (2.5 mM)-high (15 mM)-low-high-low-high followed by KCl depolarization. Images were collected during glucose challenge to generate image stacks that were used to quantify the calcium signal at the population and single cell level over time. Representative images of the peak calcium response to low glucose, high glucose, and KCl for a mouse islet are shown in Fig 1B. Fluo-4 dye penetrates the first and second cellular layers of intact islets [26]. The fluorescent signal from cells at the edge of the islets (15 mM glucose and KCl treatments, Fig 1B) appears brighter because cells in the middle, located in the z-plane, are not in focus.


Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression.

Kenty JH, Melton DA - PLoS ONE (2015)

A rise in intracellular calcium corresponds to insulin secretion.(A) Overview of calcium imaging method for islets: intact islets were plated on a 96 well plate. Islets were stained with the Fluo-4 AM and then washed in fasting solution. Calcium influx of the population of cells in the islet or single cells was imaged following the addition of glucose. Same islets were imaged again after fixation and immunofluorescence staining with INS, GCG, and NKX6.1. (B) Representative images of WT mouse islets after stimulating with 2.5 and 15 mM glucose followed by 30 mM KCl. (C) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity for WT mouse islets shown in Fig 1B (7 islets) with corresponding S1 Movie. The calcium influx response for each mouse islet was normalized to the starting fluorescence intensity data point during the initial low glucose incubation. The normalized fluorescence intensities of 7 WT mouse islets were averaged and plotted on the y-axis with standard deviations. Islets were challenged sequentially with 2.5, 15, 2.5, 15, 2.5, and 15 mM glucose and 30 mM KCl. Fluorescence was measured at 126 time points throughout the series of glucose challenges, normalized to the starting fluorescence intensity, and averaged across all islets at each time point. The standard deviation at each time point ranged between ± 2 to 8 a.u.c (area under the curve). The x-axis represents time (in seconds). P-value was calculated from the difference between ave a.u.c. during low glucose and high glucose stimulations. Significance of calcium influx response was labeled * when the P-value was between 0.05 and 0.001 and ** when the P-value was below 0.001. (D) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity of a total of eight WT mouse islets with corresponding S2 Movie. Population measurements of dynamic normalized Fluo-4 fluorescence intensity for mouse islets is shown in purple, and the ELISA measurements of secreted mouse insulin for the same batch of islets is shown in blue. Challenges were done with 5 minutes of 2.5 mM, 25 minutes of 15 mM, and 5 minutes of 30 mM KCl.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390334&req=5

pone.0122044.g001: A rise in intracellular calcium corresponds to insulin secretion.(A) Overview of calcium imaging method for islets: intact islets were plated on a 96 well plate. Islets were stained with the Fluo-4 AM and then washed in fasting solution. Calcium influx of the population of cells in the islet or single cells was imaged following the addition of glucose. Same islets were imaged again after fixation and immunofluorescence staining with INS, GCG, and NKX6.1. (B) Representative images of WT mouse islets after stimulating with 2.5 and 15 mM glucose followed by 30 mM KCl. (C) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity for WT mouse islets shown in Fig 1B (7 islets) with corresponding S1 Movie. The calcium influx response for each mouse islet was normalized to the starting fluorescence intensity data point during the initial low glucose incubation. The normalized fluorescence intensities of 7 WT mouse islets were averaged and plotted on the y-axis with standard deviations. Islets were challenged sequentially with 2.5, 15, 2.5, 15, 2.5, and 15 mM glucose and 30 mM KCl. Fluorescence was measured at 126 time points throughout the series of glucose challenges, normalized to the starting fluorescence intensity, and averaged across all islets at each time point. The standard deviation at each time point ranged between ± 2 to 8 a.u.c (area under the curve). The x-axis represents time (in seconds). P-value was calculated from the difference between ave a.u.c. during low glucose and high glucose stimulations. Significance of calcium influx response was labeled * when the P-value was between 0.05 and 0.001 and ** when the P-value was below 0.001. (D) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity of a total of eight WT mouse islets with corresponding S2 Movie. Population measurements of dynamic normalized Fluo-4 fluorescence intensity for mouse islets is shown in purple, and the ELISA measurements of secreted mouse insulin for the same batch of islets is shown in blue. Challenges were done with 5 minutes of 2.5 mM, 25 minutes of 15 mM, and 5 minutes of 30 mM KCl.
Mentions: Towards the goal of assessing the function of healthy and diseased islets, a method was developed to image calcium flux of whole islets (population analysis) and individual islet cells (single cell analysis) in intact islets followed by fixation and immunostaining to examine marker expression profiles of calcium-imaged cells (Fig 1A). Purified islets were loaded with the indicator, Fluo-4 AM, which fluoresces upon binding calcium. Islets were imaged while being stimulated with a series of sequential glucose challenges: low (2.5 mM)-high (15 mM)-low-high-low-high followed by KCl depolarization. Images were collected during glucose challenge to generate image stacks that were used to quantify the calcium signal at the population and single cell level over time. Representative images of the peak calcium response to low glucose, high glucose, and KCl for a mouse islet are shown in Fig 1B. Fluo-4 dye penetrates the first and second cellular layers of intact islets [26]. The fluorescent signal from cells at the edge of the islets (15 mM glucose and KCl treatments, Fig 1B) appears brighter because cells in the middle, located in the z-plane, are not in focus.

Bottom Line: Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression.Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells.Human islet cells were also assessed and showed functional α and β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.

No MeSH data available.