Journal: Cell reports

Article Title: Pericyte dysfunction and impaired vasomotion are hallmarks of islets during the pathogenesis of type 1 diabetes

doi: 10.1016/j.celrep.2023.112913

Figure Lengend Snippet: Living pancreas slices were incubated with Fluo4 and NG2-Alexa647. (A) Confocal image of a pericyte in an islet from a NDdonor (nPOD6516) in basal glucose concentration (3 mM), upon high glucose (11 mM) alone, or in the presence of theophylline (20 μM; 11G + theo). Scale bar: 10 μm. (B) Heatmap showing changes in Fluo4 fluorescence (DF/F; %) in pericytes in islets from ND donors elicited by 11 mM glucose and then theo (20 μM; in 11G). Each line is a different pericyte. (C) Representative traces showing changes in Fluo4 fluorescence elicited by glucose and theo. (D) Net area under the curve (AUC) of fluorescence traces for individual pericytes. *p < 0.05 (paired t test; n = 113 pericytes from 5 ND donors). (E) Heatmaps showing changes in Fluo4 fluorescence in pericytes from ND (n = 9), Aab+ (n = 7), and T1D donors (n = 6). Donors are separated by white dashed lines. T1D donor data are presented based on T1D duration, while data from ND and Aab+ donors are presented according to . (F) Traces showing average changes in Fluo4 fluorescence elicited 11mM glucose for all islet pericytes from each group. Average (thicker lines) ± SEM values are shown. (G) Net AUC of fluorescence traces during stimulation with 11G (dots: data point for each donor, bars: mean ± SEM). For each donor, responses from around 3–36 pericytes were recorded, and an average value calculated. *p < 0.05 (one-sample t test, theoretical mean = 0).

Article Snippet: Living human pancreas slices were incubated with the cytosolic calcium indicator ([Ca 2+ ]i) Fluo4-AM (6 mM, Invitrogen, cat. nr. F14201) in 3 mM glucose solution prepared in HEPES buffer (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl 2 , 1 mM MgCl2, 25 mM HEPES, 0.1% BSA [w/v], pH 7.4), supplemented with aprotinin (25 KIU), at room temperature and in the dark with either (a) DyLight 649 lectin from Lycopersicon Esculentum (3.3 mg/mL, VectorLabs, cat. nr. DL1178) for 1 hour; or with (b) a fluorescent-conjugated antibody against the pericyte marker neuron-glial antigen 2 (NG2-alexa647; 1:50, R&D Systems, cat. nr. Fab2585R) for 2 hours.

Techniques: Incubation, Concentration Assay, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Identification of hOPC markers. a Three markers of hOPCs were identified using cell immunofluorescence staining: PDGFR-α, A2B5, and NG2. Scale bar is 200 μm. b Images of single cells detached in the presence of PDGFR-α+, A2B5+, and NG2+ cells, as observed using flow cytometry. Ch01: Bright field, Ch03: A2B5-PE, Ch07: PDGFR-α-BV421, Ch11: NG2-APC. Scale bar is 20 μm. c Gating strategy to define PDGFR-α/A2B5/NG2 cells. R4: PDGFR-α+ cells, R5: A2B5+ cells, R2: NG2+ cells. d scRNA-seq of hOPCs. Maps of t-SNE of 7885 cells from high-dimensional images of hOPCs coloured according to the cell type. hOPC human oligodendrocyte progenitor cells, PDGFR-α platelet-derived growth factor receptor alpha, scRNA-seq single-cell RNA sequencing

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, RNA Sequencing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: The expression of PDGFR-α in positive and negative cells. a Gating strategy to define PDGFR-α+ cells in A2B5+/− and NG2+/− populations. b Images of single cells detached in the presence of A + P +, A − P +, N + P +, and N − P + cells. c , d Cell counts of different cell populations. Bars represent the mean. Error bars show the standard error of the mean. A + P + , PDGFR-α+ cells in A2B5+ cells; A − P + , PDGFR-α+ cells in A2B5− cells; N + P + , PDGFR-α+ cells in NG2+ cells; N − P + , PDGFR-α+ cells in NG2− cells

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Differential expression analysis. a Volcano plot illustrating differentially regulated gene expression between the four groups of cells. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. Values are presented as the log2 of tag counts. b Heat map showing the relative gene expression in NG2+/− and A2B5+/− cells. Red and green indicate upregulated and downregulated genes, respectively. c , d Graph showing the FPKM value of markers in different cell populations. Bars represent the mean. Error bars show the standard error of the mean. FPKM fragments per kilobase of exon model per million mapped fragments

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Quantitative Proteomics, Gene Expression