Journal: bioRxiv

Article Title: HNF1α transcriptional activation and repression maintain human islet α and β cell function

doi: 10.1101/2022.09.25.509394

Figure Lengend Snippet: RNA-seq of HNF1AKD β cells shows that HNF1α regulates insulin secretion, metabolism, developmental pathways, and cell-cell signaling in β cells. (A) Schematic of FACS scheme for isolation of transduced live β cells (HPi2+GFP+NTPDase3+) from control and HNF1AKD pseudoislets for downstream RNA sequencing. (B) HNF1A transcripts per million (TPM) in sequenced samples. (C) Differential expression (DE) analysis revealed significantly up- and down-regulated genes after HNF1AKD in β cells; thresholds: Fold change (FC)= 1.5, adjusted P -value= 0.05. (D) Heatmap of DEGs in β cells after HNF1AKD. Significantly (E) downregulated and (F) upregulated gene ontology (GO) pathways and (G) downregulated KEGG pathways in HNF1AKD relative to control β cells. P = Benjamini-Hochberg adjusted P -value; all P <0.05.

Article Snippet: Then, cells were stained with the following primary-secondary conjugated antibodies: HPi2-PE/Cy7 (Novus Biologicals NBP1-18946PECY7), NTPDase3-647 (clone hN3-B3S; Jean Sévigny Lab) and CD26-PE (BioLegend 302706).

Techniques: RNA Sequencing, Isolation, Control, Quantitative Proteomics

Journal: bioRxiv

Article Title: HNF1α transcriptional activation and repression maintain human islet α and β cell function

doi: 10.1101/2022.09.25.509394

Figure Lengend Snippet: RNA-seq of HNF1AKD α cells identifies dysregulation of calcium channel complexes and ATPase-coupled transmembrane transport, as well as hormone secretion pathways shared with β cells. (A) Schematic of methods for isolation of transduced α cells (HPi2+GFP+CD26+) from control and HNF1AKD pseudoislets for downstream RNA sequencing; (i) brightfield and (ii) blue light (488nm) images of human HNF1AKD pseudoislets, scale bars=1000μm. (B) HNF1A transcripts per million (TPM) in sequenced α cell samples. (C) Differential expression (DE) analysis revealed significantly up- and down-regulated genes after HNF1AKD in α cells; thresholds: FC= 1.5, adjusted P -value= 0.05. (D) Venn diagram comparing α versus β cell DEGs revealed shared and cell-specific consequences of HNF1AKD. Gene ontology (GO) pathways of (E) shared and (F) α cell enriched DEG sets. (G) Boxplots displaying TPM of select DEGs. P = Benjamini-Hochberg adjusted P -value; * P <0.05; all P <0.05 for GO pathways.

Article Snippet: Then, cells were stained with the following primary-secondary conjugated antibodies: HPi2-PE/Cy7 (Novus Biologicals NBP1-18946PECY7), NTPDase3-647 (clone hN3-B3S; Jean Sévigny Lab) and CD26-PE (BioLegend 302706).

Techniques: RNA Sequencing, Isolation, Control, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Cryopreservation and post-thaw characterization of dissociated human islet cells

doi: 10.1371/journal.pone.0263005

Figure Lengend Snippet: (A) Representative FACS plots showing cell viability and the distribution of distinct pancreatic single islet cell populations. Islets (from donor ID# R292) were dispersed as described in the Materials and Methods, and analyzed before cryopreservation (fresh, unfrozen control). Dispersed islet cells from the same donor were cooled at 1°C/min to –40°C in the presence of 10% DMSO and 6% HES, then stored in liquid nitrogen. Cells were thawed rapidly in a 37°C water bath, and the cryoprotectants were removed by serial dilution as described in the Materials and Methods. The cells were analyzed immediately ( after w/o culture) or after overnight culture ( after w culture). The cells were first stained with LIVE/DEAD Fixable near-IR dead cell dye to assess cell viability and analyzed using specific cell surface markers to stain distinct cell populations: CD31 for endothelial cells; CD45 for leukocytes; CD90 for mesenchymal cells; EpCAM for epithelial cells including acinar, ductal and islet cells; HPx1 for acinar cells and HPi2 for islet cells. FACS analyses were able to detect most of cell populations before after cryopreservation. (B) Summary of FACS analysis of viability and EpCAM (surface marker for islet cells) expression in three pancreatic islet donors. Fresh (black bars) and cryopreserved (red bars) dispersed islet cells from donors (see ) were analyzed by flow cytometry. Cryopreserved cells were also cultured overnight and analyzed the next day (blue bars). Left panel: Cell viability is expressed as mean relative abundance ± standard error of the mean with actual data points represented as individual dots. Right panel: EpCAM expression.

Article Snippet: The following antibodies were used for FACS experiments at 1:100 (v/v) final concentration, CD31-FITC (BioLegend, 303103, San Diego, CA), CD45-PE/Dazzle594 (BioLegend, 304052), CD90-Alexa Fluor 700 (BioLegend, 328120), EpCAM-APC (BioLegend, 324208), HPi2-Alexa405 (Novus Biologicals, NBP1-18946AF405, Centennial, CO), and HPx1-PE (Novus Biologicals, NBP1-18951PE).

Techniques: Control, Serial Dilution, Staining, Marker, Expressing, Flow Cytometry, Cell Culture

Journal: Cell Reports Medicine

Article Title: Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive β Cell Destruction

doi: 10.1016/j.xcrm.2020.100097

Figure Lengend Snippet:

Article Snippet: After centrifugation, cells were resuspended in PBS with 1% BSA, and stained with 1:100 diluted A647-conjugated HPi2 antibody (Novus Biologicals NBP1-18946AF647) and DyLight 488 conjugated HPx1 Antibody (Novus Biologicals NBP1-18951G), for 30 min at 4°C.

Techniques: Virus, Recombinant, Cytotoxicity Assay, Purification, Luminex, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Labeling, Plasmid Preparation, Software

Journal: Cell reports

Article Title: ZIGIR, a Granule-Specific Zn 2+ Indicator, Reveals Human Islet α Cell Heterogeneity

doi: 10.1016/j.celrep.2020.107904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal APC-conjugated HIC1 , Novus Biologicals , NBP1–18946APC.

Techniques: Virus, Recombinant, Software, Imaging

Journal: bioRxiv

Article Title: Glucagon and GLP-1 Accelerate Pseudo-Islet Assembly and Unmask Sex-Specific Islet Fragmentation Dynamics

doi: 10.1101/2025.08.10.669547

Figure Lengend Snippet: a) Workflow: Human donor islets derived from both male and female donors were dissociated into single cells, stained with HIC1-2B4 AF488 (pan-endocrine marker), HIC1-8G12 PE (non-β-endocrine cell marker), and CD9 APC (predominantly δ-cell marker), and sorted into distinct α-, β-, and δ-cell populations via FACS. These populations were then used to form pseudo-islets either from single populations (b) and d) for α-cells ONLY and c) and e) for β-cells ONLY) or combinations thereof (f and g). Pseudo-islets were formed in ultra-low attachment 6-well plates and combined in a 1:10 ratio of endocrine to Mile Sven 1 (MS1) cells (mouse pancreatic endothelial cells). Hormone supplementation consisted of 0.1 µg/mL GCG (pink), 0.1 µg/mL GLP-1 (teal), and 100 nM somatostatin (SST, orange), compared to the control (CTRL, black), with supplements administered every other day. Growth and formation of pseudo-islets depicted in all subpanels are from 2-3 days post-seeding via EPI-light microscopy. Donors were pooled by sex; panels b), c), and f) correspond to female donors and panels d), e), and g) to male donors. Statistical analysis was done by two-way ANOVA with a Benjamini-Hochberg correction for multiple comparisons. P-values are indicated for each comparison; p < 0.05 was considered the cutoff for statistical significance.

Article Snippet: The antibodies used were: HIC1-2B4 A488 conjugated (Novus Biologicals, REF# NBP1-18946AF488) at a 1:100 dilution, HIC1-8G12 PE conjugated (provided by the Grompe lab) at 1:50 dilution, and anti-CD9 APC conjugated to APC (Thermo Fisher Invitrogen, MA1-10307) at a 1:20 dilution.

Techniques: Derivative Assay, Staining, Marker, Control, Light Microscopy, Comparison