Insulator-based dielectrophoresis-assisted separation of insulin secretory vesicles
- Department of Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, United States
- School of Molecular Sciences, Arizona State University, United States
- Department of Biological Sciences, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, United States
Figures
Figure 1 with 3 supplements
Schematic diagram for the formation of heterogenous insulin vesicles in INS-1E cells, graphical summation of disparate vesicle protein identifications, and processing of insulin vesicles including direct current insulator-based dielectrophoresis (DC-iDEP) device.
(A) Insulin vesicle formation and maturation in a pancreatic β-cell. Newly synthesized insulin is packed inside secretory vesicles which mature to store crystalline insulin in vesicles until …
Figure 1—figure supplement 1
Glucose sensitivity of INS-1E insulinoma cells was tested by stimulation at increasing concentrations of glucose and measurement of insulin secretion by enzyme linked immunosorbent assay (ELISA).
Figure 1—figure supplement 2
Selection of fractions for use in separation experiments.
(A) Fractions of the density column were screened for insulin content in the enzyme linked immunosorbent assay (ELISA) assay. (B) Western blotting of the density column fractions revealed high …
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Figure 1—figure supplement 2—source data 1
Full image of western blotting (WB) with labels indicating synaptotagmin IX, SEC16 B, and cytochrome c.
- https://cdn.elifesciences.org/articles/74989/elife-74989-fig1-figsupp2-data1-v1.pdf
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Figure 1—figure supplement 2—source data 2
Raw image of western blotting (WB).
- https://cdn.elifesciences.org/articles/74989/elife-74989-fig1-figsupp2-data2-v1.pdf
Figure 1—figure supplement 3
The identity of insulin vesicles was confirmed via several microscopy methods: confocal microscopy, transmission electron microscopy, and cryo-electron microscopy.
(A) Fluorescence confocal microscopy maximum projection image of enriched insulin vesicles. (B) Transmission electron microscopy (TEM) image of enriched insulin vesicles negatively stained with …
Figure 2
Colocalization of insulin vesicle marker used in this study (synaptotagmin IX) with insulin.
(A, E) Nuclei of INS-1E insulinoma cells stained with NucBlue. (B, F) Synaptotagmin IX labeled with rabbit anti-synaptotagmin IX and goat anti-rabbit IgG (H+L), Alexa 647. (C, G) Insulin hormone …
Figure 3
Schematic diagram of direct current insulator-based dielectrophoresis (DC-iDEP) system operated in a discovery or scanning mode.
As a function of the design of the sawtooth channel with different gate sizes along the channel, the applied voltage defines dielectrophoretic (DEP) and electrokinetic (EK) forces at each gate, and …
Figure 4
Comparison of distributions for the biophysical properties as reflected in EKMr values of n-insulin vesicles (blue circles) and g-insulin vesicles (yellow squares) with varied voltages applied.
Fluorescent intensities of captured n- and g-insulin vesicles with different EKMr values were recorded at each gate. Each data point reflects fluorescent intensities recorded at three subsequent …
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Figure 4—source data 1
The raw data depicted in Figure 4.
- https://cdn.elifesciences.org/articles/74989/elife-74989-fig4-data1-v1.xlsx
