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High-Resolution Recording of the Circadian Oscillator in Primary Mouse α - and β -Cell Culture

View Article: PubMed Central - PubMed

ABSTRACT

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

No MeSH data available.


Related in: MedlinePlus

Purity of α- and β-cell populations, separated by fluorescence-activated cell sorting (FACS) form ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets. Sorted samples of Venus- and Cherry-positive cells were subsequently analyzed by the second run of FACS. (A–C) FACS-assessed purity analysis of obtained α-cell population: (A) dot plot presenting distribution of cell size [forward scatter detector (FSC), x-axis] versus granularity [side scatter detector (SSC), y-axis] of Venus-positive cells within gated cell population; (B) dot plot showing sample enrichment for Venus-positive cells and complete absence of Cherry-positive cells; (C) DAPI-assessed analysis of α-cell viability after sorting, indicating 97% viable cells in the sample. (D–F) FACS-assessed purity analysis of obtained β-cell population: (D) dot plot presenting distribution of cell size (FSC, x-axis) versus granularity (SSC, y-axis) of Cherry-positive cells within gated cell population; (E) dot plot showing enrichment of sample for Cherry-positive cells and absence of Venus-positive contaminants; (F) DAPI-assessed analysis of β-cell viability after sorting, indicating approximately 97% viable cells in the sample. (G) Histogram representing average purity in analyzed samples (data are presented as mean ± SEM, N = 6 experiments for α-cells; and N = 10 for β-cells). (H) Representative fluorescent images illustrating sorted Venus- and Cherry-positive cell populations (α- and β-cells, respectively). Scale bar = 100 μm.
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Figure 3: Purity of α- and β-cell populations, separated by fluorescence-activated cell sorting (FACS) form ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets. Sorted samples of Venus- and Cherry-positive cells were subsequently analyzed by the second run of FACS. (A–C) FACS-assessed purity analysis of obtained α-cell population: (A) dot plot presenting distribution of cell size [forward scatter detector (FSC), x-axis] versus granularity [side scatter detector (SSC), y-axis] of Venus-positive cells within gated cell population; (B) dot plot showing sample enrichment for Venus-positive cells and complete absence of Cherry-positive cells; (C) DAPI-assessed analysis of α-cell viability after sorting, indicating 97% viable cells in the sample. (D–F) FACS-assessed purity analysis of obtained β-cell population: (D) dot plot presenting distribution of cell size (FSC, x-axis) versus granularity (SSC, y-axis) of Cherry-positive cells within gated cell population; (E) dot plot showing enrichment of sample for Cherry-positive cells and absence of Venus-positive contaminants; (F) DAPI-assessed analysis of β-cell viability after sorting, indicating approximately 97% viable cells in the sample. (G) Histogram representing average purity in analyzed samples (data are presented as mean ± SEM, N = 6 experiments for α-cells; and N = 10 for β-cells). (H) Representative fluorescent images illustrating sorted Venus- and Cherry-positive cell populations (α- and β-cells, respectively). Scale bar = 100 μm.

Mentions: In order to simultaneously label α- and β-cells within the islets, a ProGcg-Venus/RIP-Cherry/Per2:Luc reporter mouse line was established (Figure 1), allowing for the highly specific separation of nearly pure endocrine cell populations. To this end, following islet isolation and gentle trypsinization, dispersed cells were sorted by FACS based on cellular fluorescence characteristics, cell size (based on the data of Forward Scatter detector, FSC), and granularity (based on the data of Side Scatter detector, SSC; see Figures 2A,B). Of note, Cherry-positive cells showed higher cell granularity than Venus-positive cells (compare two histograms in Figure 2B). In parallel, cell viability was assessed by utilizing DRAQ7™, a dye that binds to DNA when cell membrane permeability is altered after initiation of cell death. Overall viability across preparations was near 90% (Figure 2C), with almost a 10-fold higher percentage of cell death for Cherry-positive cells (up to 20%) than for Venus-positive cells (Figures 2D,E), suggesting a higher sensitivity of Cherry-positive cells to the islet isolation, trypsinization, and/or sorting processes. The average number of harvested viable Cherry-positive cells per mouse was 39,292 ± 6,887, which is more than threefold higher than for Venus-positive cells (12,521 ± 1,885) (Figure 2F), reflecting the physiological ratio between these two cell types within mouse islets (22). In addition, the purity of the obtained Venus- and Cherry-positive cell populations was assessed by a complementary round of FACS analysis (Figures 3A–G). According to this analysis, the α-cell population comprises more than 90% viable Venus-positive cells without detectable contamination with Cherry-positive cells (Figures 3A–C,G), while the β-cell population contained up to 96% viable Cherry-positive cells without visible Venus-positive contaminants (Figures 3D,E,G). Finally, the morphological examination of sorted cell populations with a fluorescent microscope confirmed their high purity (Figure 3H).


High-Resolution Recording of the Circadian Oscillator in Primary Mouse α - and β -Cell Culture
Purity of α- and β-cell populations, separated by fluorescence-activated cell sorting (FACS) form ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets. Sorted samples of Venus- and Cherry-positive cells were subsequently analyzed by the second run of FACS. (A–C) FACS-assessed purity analysis of obtained α-cell population: (A) dot plot presenting distribution of cell size [forward scatter detector (FSC), x-axis] versus granularity [side scatter detector (SSC), y-axis] of Venus-positive cells within gated cell population; (B) dot plot showing sample enrichment for Venus-positive cells and complete absence of Cherry-positive cells; (C) DAPI-assessed analysis of α-cell viability after sorting, indicating 97% viable cells in the sample. (D–F) FACS-assessed purity analysis of obtained β-cell population: (D) dot plot presenting distribution of cell size (FSC, x-axis) versus granularity (SSC, y-axis) of Cherry-positive cells within gated cell population; (E) dot plot showing enrichment of sample for Cherry-positive cells and absence of Venus-positive contaminants; (F) DAPI-assessed analysis of β-cell viability after sorting, indicating approximately 97% viable cells in the sample. (G) Histogram representing average purity in analyzed samples (data are presented as mean ± SEM, N = 6 experiments for α-cells; and N = 10 for β-cells). (H) Representative fluorescent images illustrating sorted Venus- and Cherry-positive cell populations (α- and β-cells, respectively). Scale bar = 100 μm.
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Related In: Results  -  Collection

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Figure 3: Purity of α- and β-cell populations, separated by fluorescence-activated cell sorting (FACS) form ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets. Sorted samples of Venus- and Cherry-positive cells were subsequently analyzed by the second run of FACS. (A–C) FACS-assessed purity analysis of obtained α-cell population: (A) dot plot presenting distribution of cell size [forward scatter detector (FSC), x-axis] versus granularity [side scatter detector (SSC), y-axis] of Venus-positive cells within gated cell population; (B) dot plot showing sample enrichment for Venus-positive cells and complete absence of Cherry-positive cells; (C) DAPI-assessed analysis of α-cell viability after sorting, indicating 97% viable cells in the sample. (D–F) FACS-assessed purity analysis of obtained β-cell population: (D) dot plot presenting distribution of cell size (FSC, x-axis) versus granularity (SSC, y-axis) of Cherry-positive cells within gated cell population; (E) dot plot showing enrichment of sample for Cherry-positive cells and absence of Venus-positive contaminants; (F) DAPI-assessed analysis of β-cell viability after sorting, indicating approximately 97% viable cells in the sample. (G) Histogram representing average purity in analyzed samples (data are presented as mean ± SEM, N = 6 experiments for α-cells; and N = 10 for β-cells). (H) Representative fluorescent images illustrating sorted Venus- and Cherry-positive cell populations (α- and β-cells, respectively). Scale bar = 100 μm.
Mentions: In order to simultaneously label α- and β-cells within the islets, a ProGcg-Venus/RIP-Cherry/Per2:Luc reporter mouse line was established (Figure 1), allowing for the highly specific separation of nearly pure endocrine cell populations. To this end, following islet isolation and gentle trypsinization, dispersed cells were sorted by FACS based on cellular fluorescence characteristics, cell size (based on the data of Forward Scatter detector, FSC), and granularity (based on the data of Side Scatter detector, SSC; see Figures 2A,B). Of note, Cherry-positive cells showed higher cell granularity than Venus-positive cells (compare two histograms in Figure 2B). In parallel, cell viability was assessed by utilizing DRAQ7™, a dye that binds to DNA when cell membrane permeability is altered after initiation of cell death. Overall viability across preparations was near 90% (Figure 2C), with almost a 10-fold higher percentage of cell death for Cherry-positive cells (up to 20%) than for Venus-positive cells (Figures 2D,E), suggesting a higher sensitivity of Cherry-positive cells to the islet isolation, trypsinization, and/or sorting processes. The average number of harvested viable Cherry-positive cells per mouse was 39,292 ± 6,887, which is more than threefold higher than for Venus-positive cells (12,521 ± 1,885) (Figure 2F), reflecting the physiological ratio between these two cell types within mouse islets (22). In addition, the purity of the obtained Venus- and Cherry-positive cell populations was assessed by a complementary round of FACS analysis (Figures 3A–G). According to this analysis, the α-cell population comprises more than 90% viable Venus-positive cells without detectable contamination with Cherry-positive cells (Figures 3A–C,G), while the β-cell population contained up to 96% viable Cherry-positive cells without visible Venus-positive contaminants (Figures 3D,E,G). Finally, the morphological examination of sorted cell populations with a fluorescent microscope confirmed their high purity (Figure 3H).

View Article: PubMed Central - PubMed

ABSTRACT

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

No MeSH data available.


Related in: MedlinePlus