Journal: Science Advances

Article Title: 3D bioprinting of collagen-based high-resolution internally perfusable scaffolds for engineering fully biologic tissue systems

doi: 10.1126/sciadv.adu5905

Figure Lengend Snippet: ( A ) Schematic design and 3D printer machine pathing G-code of a dual parallel channel multi-material CHIPS with pancreatic vascular cell bioink (MIN6, HUVEC, and MSC) regions surrounding both sides of the channels. ( B ) Pancreatic CHIPS FRESH printed and visualized via bright-field stereomicroscope (inset) and 3D confocal fluorescence imaging of the optically cleared pancreatic scaffold after 12 days of static culture. ( C ) XY midplane view from confocal fluorescence image of the 12-day statically cultured pancreatic CHIPS following 3D vascular network segmentation for quantification of network diameter and density within the migratory zones. ( D ) Example confocal fluorescence images revealing additional cell migration into the acellular regions of the CHIPS beneath the cellular regions guided by the printed collagen filaments. ( E ) XY midplane projection view from 3D confocal fluorescence imaging of the optically cleared 12-day VAPOR perfused pancreatic CHIPS. ( F ) Graphic illustration and example ROIs depicting evidence of early MIN6 pancreatic bud and microlumen formation with actin (green) and insulin (magenta) fluorescence images from ROIs 2 and 3 in (E). ( G ) Graphic illustration and quantification for insulin secretion ELISA assay from 1.5-hour glucose-stimulated [ratio of high glucose (HG) to low glucose (LG)] insulin secretion experiment between 12-day static and perfusion cultured pancreatic CHIPS (means ± SD; ** P < 0.01 for N = 3 static tissues; N = 2 perfused tissues, unpaired t test).

Article Snippet: MIN6 (mouse insulinoma cell line) (Addexbio, C0018008) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 11-965-092) with 15% fetal bovine serum (v/v), 2 mM sodium pyruvate, 20 mM Hepes, and 0.05 mM β-mercaptoethanol, using passages 9 to 15.

Techniques: Fluorescence, Imaging, Cell Culture, Migration, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Optimised proteomic analysis of insulin granules from MIN6 β-cells identifies Scamp3, a novel regulator of insulin secretion and content

doi: 10.1101/2024.04.23.590838

Figure Lengend Snippet: ( A ) Optiprep workflow : MIN6 post-nuclear supernatant is loaded atop 5 fixed concentrations of Optiprep and ultra-centrifuged for 75min at 100,000g. ( B ) Representative example of visible subcellular fractionation distribution after ultra-centrifugation. ( C ) Representative quantification of insulin enrichment from 16 fractions of Optiprep by insulin ELISA ( D ) Western blot analysis of pro- and insulin enrichment of Optiprep fractions, as well as marker proteins for subcellular components of beta-cells. ( E ) Percoll workflow : Fractions 11 and 12 from Optiprep gradients following insulin enrichment analysis are loaded on top of 27% Percoll and ultra-centrifuged for 60min at 35,000g. ( F ) SDS-PAGE analysis of insulin enrichment of Percoll fractionation, as well as marker proteins for subcellular components of MIN6 beta-cells.

Article Snippet: The MIN6 mouse insulinoma β-cells were purchased from AddexBio TM and cultured in standard culture media as stated previously .

Techniques: Fractionation, Centrifugation, Enzyme-linked Immunosorbent Assay, Western Blot, Marker, SDS Page

Journal: bioRxiv

Article Title: Optimised proteomic analysis of insulin granules from MIN6 β-cells identifies Scamp3, a novel regulator of insulin secretion and content

doi: 10.1101/2024.04.23.590838

Figure Lengend Snippet: ( A ) Confocal fluorescence imaging of MIN6 cells labelled with anti-insulin co-stained with anti-ZIP8 (n = 3), anti-Synaptophysin (n = 3), anti-Synaptotagmin-13 (n = 4), anti-Syntaxin-7 (n = 3), anti-Scamp3 (n = 3) and anti-Chromogranin A (n = 3). Enrichment profile of candidate proteins across 12 fractions from LC-MS/MS analysis. ( B ) Quantification of colocalization of candidate proteins with insulin using Pearson’s correlation coefficient (with Costes’ automatic thresholds). Filled dots in purple represent the number of experiments (Run #) these proteins appear within pRoloc analyses. ( C ) Confocal fluorescence imaging of human islets labelled with anti-insulin, co-stained with anti-synaptophysin (n = 1), anti-synaptotagmin-13 (n = 1), anti-syntaxin7 (n = 1) and anti-Scamp3 (n = 1). ( D ) Quantification of colocalization of candidate proteins with insulin from individual islets from a single human non-type 2 diabetic donor for each protein using Pearson’s correlation coefficient (with Costes’ automatic thresholds). All error bars represent standard error of the mean (+-S.E.M.)

Article Snippet: The MIN6 mouse insulinoma β-cells were purchased from AddexBio TM and cultured in standard culture media as stated previously .

Techniques: Fluorescence, Imaging, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Optimised proteomic analysis of insulin granules from MIN6 β-cells identifies Scamp3, a novel regulator of insulin secretion and content

doi: 10.1101/2024.04.23.590838

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of INS-1 cell lysates 48 hours post transfection with a control non-targeting siRNA and two unique siRNAs specific to Scamp3 (siScamp3#1 and siScamp3#2) in 3 experimental replicates together (n = 3). ( B ) Densitometry quantification of knockdown efficiency of Scamp3 48 hours post transfection (n = 3) normalized to GAPDH. ( C ) Insulin SDS-PAGE analysis of INS-1 cell lysates 48 hours post transfection of control and siRNA KD INS-1 cells. ( D ) Densitometry quantification of insulin content in control and siRNA KD INS-1 cells (n = 3) normalized to beta-actin. ( E ) Glucose-stimulated insulin secretion assay: insulin concentration after basal (2.8 mM) and stimulation (16.7 mM) glucose conditions from INS-1 control and siRNA KD cells measured by insulin HTRF (n = 8). ( F ) Fold-change of basal to stimulation glucose conditions from E (n = 8). ( G ) Representative confocal fluorescence imaging of human non-type 2 diabetic (n = 5) and type 2 diabetic (n = 5) islets labelled with anti-insulin and co-stained with Scamp3. ( H ) Mean fluorescence intensity of Scamp3 within β-cells of human non-type 2 diabetic and type 2 diabetic islets ( I ) Quantification of colocalization of Scamp3 with insulin using Pearson’s correlation coefficient (with Costes’ automatic thresholds) in human non-type 2 diabetic and type 2 diabetic islets. All error bars represent standard error of the mean (+-S.E.M.)

Article Snippet: The MIN6 mouse insulinoma β-cells were purchased from AddexBio TM and cultured in standard culture media as stated previously .

Techniques: SDS Page, Transfection, Concentration Assay, Fluorescence, Imaging, Staining