A high-throughput assay for the measurement of Ca2+ oscillations and insulin release from uniformly sized mouse β-cell (MIN6) spheroids
- Department of Cellular and Molecular Medicine, KU Leuven, Belgium
- Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Neuroscience, Belgium
- 4Dcell, France
Figures
Figure 1
Hydrogel design.
(A) Schematic top-view representation of the hydrogel, illustrating a honeycomb arrangement of 19 microwells per gel. (B) Schematic cross-sectional depiction of the microwells (not to scale). (C) Computer-aided design (CAD) rendering of the hydrogel positioned within the well of a glass-bottom 96-well plate. (D) Entire glass-bottom 96-well plate with microstructured polyethylene glycol (PEG)-based hydrogel in each well. (E) Microwell with mouse embryonic fibroblasts attached to the contact point, which is a result of the molds being in contact with the glass bottom during polymerization (top). Mean contact point diameter for two independently moulded samples. N=16 wells (304 contact points) per sample. Statistical difference was analyzed using one-way ANOVA and Tukey’s post hoc test, where ns = not significant.
-
Figure 1—source data 1
Contact point diameters per sample.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig1-data1-v1.xlsx
Figure 2
Cell seeding and data acquisition.
(A) Schematic representation of protocol timeline. 200 μL of the mouse insulinoma 6 (MIN6) pancreatic β-cell suspension is added to each well of the 96-well plate at hour 0. Over the next 16 hr, the cells undergo sedimentation into the microwells and spontaneously self-assemble into spheroids. Two hours prior to data acquisition, 10 μM of Cal-520 is introduced for 1 hr, followed by washing and incubation in a low-glucose KREBS solution for 30 min (hours 17–18). At hour 19, the plates are transferred to the μCELL FDSS for fluorescence-based data acquisition. (B) Representative images of MIN6 β-cells following sedimentation within a microwell, captured 10 min post-seeding (top) and 16 hr post-seeding (bottom). (C) Representative fluorescence image of a section of a 96-well plate containing the Cal-520 AM-loaded MIN6 β-cell spheroids acquired using the µCELL FDSS fluorescence plate imager, distinctly showing individual spheroids arranged in a honeycomb pattern within the microwell array. (D) Histogram showing the number of spheroids still present per well at the time of the control stimulus. N=48 wells on two independent occasions.
-
Figure 2—source data 1
Number of spheroids still present per well at the time of the control stimulus.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Oscillatory behavior of Ca2+ signal is modulated by extracellular glucose concentrations.
(A) Representative Ca2+ response of a single 3D mouse insulinoma 6 (MIN6) β-cell spheroid to an increase in extracellular glucose concentration (20 mM), highlighting the visual representations of the peaks and the area under the peaks. The spheroids were initially exposed to basal extracellular glucose levels (3 mM) for 10 min, followed by an increase in extracellular glucose (20 mM) for 30 min. The protocol ended with a control stimulus of 50 mM KCl. (B) Mean frequency and the normalized area under the peaks of the fluorescent signal during the 30 min of exposure to increased concentrations of extracellular glucose. Peak threshold was set to three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N>50 spheroids per concentration. Signal values for each spheroid were normalized to the amplitude of the control stimulus. (C) Overview of Ca2+ responses of each individual MIN6 β-cell spheroid within a single well to an increase in extracellular glucose concentration (20 mM). To preserve the absolute response dynamics and inter-spheroid variability, no baseline correction was applied to the displayed traces. Gray lines represent individual spheroids, while the black line denotes the mean response across all spheroids. (D) Comparison of the mean Ca2+ fluorescence response of all individual MIN6 β-cell spheroids within a single-well (solid line) and 2D-cultured MIN6 cells (dashed line) following stimulation with 20 mM extracellular glucose.
-
Figure 3—source data 1
Source data for Figure 3A, B, C and D.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Ca2+ response to glucose of mouse insulinoma 6 (MIN6) cells in 2D culture.
(A) Characteristic Ca2+ responses of wells containing 2D MIN6 β-cell monolayers exposed to a range of extracellular glucose concentrations. The monolayers were initially exposed to basal extracellular glucose levels (3 mM) for 10 min, followed by an increase in extracellular glucose for 30 min. Between 40 and 50, the liquid dispensing head was used to aspirate supernatant from the wells. The protocol ended with a control stimulus of 50 mM KCl. (B) The number of peaks and the normalized area under the peaks of the fluorescent signal during the 30 min of exposure to increased concentrations of extracellular glucose. Peak threshold was set to three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N=8 wells.
Figure 4
KATP-agonist diazoxide inhibits oscillatory response to extracellular glucose.
(A) Characteristic Ca2+ responses to 10 mM extracellular glucose concentrations of individual mouse insulinoma 6 (MIN6) β-cell spheroids with and without 30 min of prior exposure to 100 μM diazoxide. The spheroids were initially exposed to basal extracellular glucose levels for 5 min after which the spheroids were exposed to varying concentrations of diazoxide for 30 min, followed by a 30 min period of extracellular glucose concentrations of 10 mM until a control stimulus of 50 mM KCl. Each signal was normalized over the amplitude of the control stimulus. (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 30 min of exposure to increased concentrations of extracellular glucose. Peak threshold was set to three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N>40 spheroids per concentration.
-
Figure 4—source data 1
Source data for Figure 4 A & B.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig4-data1-v1.xlsx
Figure 5
KATP-antagonists induce Ca2+ oscillatory behavior with distinct potencies.
(A) Characteristic Ca2+ responses of individual mouse insulinoma 6 (MIN6) β-cell spheroids to 10 mM of extracellular glucose with and without prior exposure to 100 nM glimepiride. The spheroids were initially exposed to basal extracellular glucose levels for 5 min after which the spheroids were exposed to varying levels of glimepiride for 30 min, followed by a 30 min period of glimepiride+extracellular glucose concentrations of 10 mM until a control stimulus of 50 mM KCl. Each signal was normalized over the amplitude of the control stimulus. (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 30 min of exposure to various concentrations of glimepiride, nateglinide, and tolbutamide. Peak threshold was set to three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N>50 spheroids per concentration.
-
Figure 5—source data 1
Source data for Figure 5 A & B.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig5-data1-v1.xlsx
Figure 6
Glimepiride and diazoxide impact glucose-dependent insulin release by mouse insulinoma 6 (MIN6) spheroids.
(A) Characteristic Ca2+ responses of individual MIN6 β-cell spheroids. The spheroids were initially exposed to basal extracellular glucose levels for 10 min after which the spheroids were exposed to either 8 mM of extracellular glucose or 8 mM of extracellular glucose supplemented with 10 nM glimepiride or 10 µM diazoxide, followed by a control stimulus of 50 mM KCl. Each signal is normalized over the maximum value of the control stimulus. (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 60 min of exposure to the various modulators. Peak threshold was set to three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N>50 spheroids per concentration. (C) Corresponding insulin concentrations present in the supernatant of the wells at the 1 hr timepoint quantified using ELISA. Data is presented as means ± SEM (n=4 wells per condition). Statistical difference was analyzed using one-way ANOVA and Tukey’s post hoc test, where ns = not significant, **p<0.01, ****p<0.0001.
-
Figure 6—source data 1
Source data for Figure 4 A, B & C.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig6-data1-v1.xlsx
Figure 7
Pregnenolone sulfate (PS) induces Ca2+-oscillatory behavior and increases insulin secretion.
(A) Characteristic Ca2+ responses of individual mouse insulinoma 6 (MIN6) β-cell spheroids exposed to an extracellular glucose concentration of 8 mM, with and without 50 μM PS. Spheroids were initially exposed to a baseline extracellular glucose concentration of 3 mM for 10 min, followed by a 1 hr incubation with varying glucose concentrations, with or without 50 μM PS. After 60 min, 15 μL of supernatant was collected for insulin level analysis, concluding with a control stimulus of 50 mM KCl. (B) Quantitative analysis of Ca2+ oscillation parameters. The frequency and normalized area under the peaks were analyzed for spheroids exposed to glucose concentrations of 3 mM, 8 mM, and 20 mM, with or without 50 μM PS. Peak threshold was set at three times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. (C) Corresponding insulin concentrations present in the supernatant of the wells at the 1 hr timepoint quantified using ELISA. Data is presented as means ± SEM (n=4 wells per condition). (D) Characteristic smoothed Ca2+ responses of individual MIN6 β-cell spheroids exposed to an extracellular glucose concentration of 8 mM+50 μM PS, with and without 10 μM isosakuranetin (ISN). Spheroids were initially exposed to a baseline extracellular glucose concentration of 3 mM for 5 min, followed by a 1 hr incubation with 8 mM of extracellular glucose+50 μM PS with and without 10 μM ISN. After 60 min, 15 μL of supernatant was collected for insulin level analysis, concluding with a control stimulus of 50 mM KCl. (E) Quantitative analysis of Ca2+ oscillation parameters. The frequency and normalized area under the peaks were analyzed for spheroids exposed to a glucose concentration of 8 mM with or without 50 μM PS, supplemented with or without 10 μM ISN. Peak threshold was set at five times the signal’s standard deviation during the initial 10 min of exposure to basal levels of extracellular glucose. (F) Corresponding insulin concentrations present in the supernatant of the wells at the 1 hr timepoint quantified using ELISA. Data is presented as means ± SEM (n=4 wells per condition). Statistical difference was analyzed using one-way ANOVA (E and F) or two-way ANOVA (B and C) and Tukey’s post hoc test, where ns = not significant, **p<0.01, ***p<0.001, ****p<0.0001.
-
Figure 7—source data 1
Source data for Figure 7 A, B, C, D, E & F.
- https://cdn.elifesciences.org/articles/110428/elife-110428-fig7-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | MIN6 | Merck | SCC623; RRID:CVCL_0431 | |
| Commercial assay or kit | Mercodia Ultrasensitive Mouse Insulin ELISA kit | Mercodia AB | 10-1249-01 RRID:AB_3101981 | |
| Chemical compound, drug | Cal-520 AM | AAT Bioquest, USA | 21130 RRID:SCR_021130 | |
| Chemical compound, drug | Diazoxide | Sigma-Aldrich | D9035 RRID:SCR_025590 | |
| Chemical compound, drug | Glimepiride | Sigma-Aldrich | PHR1617 RRID:SCR_025556 | |
| Chemical compound, drug | Nateglinide | Sigma-Aldrich | N3538 RRID:SCR_008988 | |
| Chemical compound, drug | Tolbutamide | Sigma-Aldrich | T0891 | |
| Chemical compound, drug | Pregnenolone sulfate | Sigma-Aldrich | P162 | |
| Chemical compound, drug | Isosakuranetin | Sigma-Aldrich | PHL82569 RRID:SCR_008988 | |
| Software, algorithm | Fiji | National Institutes of Health, USA | ImageJ 1.52i; RRID:SCR_002285 |
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A high-throughput assay for the measurement of Ca2+ oscillations and insulin release from uniformly sized mouse β-cell (MIN6) spheroids
eLife 15:RP110428.
https://doi.org/10.7554/eLife.110428.3
