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

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 2. Administration of APB5 induced pericytes to undergo apoptosis. A, A schematic showing the strategy of in vivo adminis- tration of anti-PDGFRb antibodies (APB5). B, Representative immunostaining for CD31 (green), NG2 (magenta), and PDGFRb (top) or PDGFRa (bottom; cyan) of the E14 cerebral wall. In top figures, white arrowhead indicates NG21PDGFRb 1 pericytes and yellow arrowhead indicates NG21PDGFRb – OPCs. In bottom figures, white arrowhead indicates NG21PDGFRa– pericytes and yellow arrowhead indicates NG21PDGFRa1 OPCs. C, Immunostaining for NG2 (green), CD31 (magenta), and DAPI (blue) in the E14 cerebral wall. D, A graph comparing the proportion of pericyte coverage, which was calculated by dividing the merged area of NG2 and CD31 by the total CD311 area in the pallium (not including the meninges), between the saline-treated, isotype control antibody-treated, and APB5-treated brains (two-sided Steel–Dwass test; the average value of four sections from each animal is plotted; N = 6 mice; p= 1.000 and p=0.011; left to right). Data are presented as the mean value 6 SD. E, Immunostaining for Cl-Casp3 (green), CD31 (magenta), and DAPI (blue) in the cerebral wall of the mice treated with antibodies or saline. The white broken line indicates the ap- ical surface. Scale bar: 50mm (B) or 100mm (C, E).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vivo, Immunostaining, Saline, Control

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 6. An in vitro coculture showed that pericytes promoted microglial proliferation. A, FACS analysis of pallial cells collected from E14 mice to detect PDGFRb , NG2, and PDGFRa expres- sion. B, Pericyte sorting strategy by FACS. NG21PDGFRa– cells were collected from E14 cerebral wall cells. C, A schematic showing the bilayer culture of CD11b1 microglia with or without NG21PDGFRa– cells (pericytes) using the Transwell insert. D, Immunostaining for CX3CR1 and Ki67 in microglia cultured with or without NG21PDGFRa cells (pericytes). E, A bar graph com- paring the density of CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). F, A bar graph comparing the proportion of Ki671 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). G, Immunostaining for CX3CR1 and BrdU in microglia cultured with or without NG21PDGFRa cells (pericytes). Scale bar: 20mm. H, A bar graph comparing the proportion of BrdU1 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). Data are pre- sented as the mean value 6 SD. Scale bar: 50 mm (D) or 20mm (G).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vitro, Immunostaining, Cell Culture, MANN-WHITNEY

Journal: PloS one

Article Title: Pro-inflammatory cytokines reduce the proliferation of NG2 cells and increase shedding of NG2 in vivo and in vitro.

doi: 10.1371/journal.pone.0109387

Figure Lengend Snippet: Figure 2. Proliferation of NG2+ cells in response to systemic lipopolysaccharide treatment. Unaltered number of NG2+/ki67+ cell numbers was found in the medial prefrontal cortex (mPFC) (A), the basolateral nucleus (BL) (B), the dentate hilus (Hilus) (C) and the molecular layer (ML) (D) 2 h after lipopolysaccharides (LPS) treatment compared to saline treatment (Saline). In contrast, a significant decreased number of NG2+/ ki67+ cells were detected in the BL (B), the Hilus (C) and the ML (D), but not in the mPFC (A), 24 h after LPS treatment compared to saline treatment. The number of Ki67+ cell in the subgranular zone (SGZ) of the hippocampal granular cell layer (E) was also unaltered after 2 h, but significantly reduced 24 h after LPS compared to saline treatment. *** indicates a significant difference at p,0.001 level. ** indicates a significant difference at p, 0.01 level. * indicates a significant difference at p,0.05 level. Images in (F–H) demonstrate a double immunoflourescence staining with antibodies directed against the endogenous proliferation marker Ki67 (F) and NG2 (G). Images (F) and (G) are superimposed in (H). Scale bar = 10 mm. doi:10.1371/journal.pone.0109387.g002

Article Snippet: Recombinant rat NG2 (kind gift from Dr William Stallcup) or recombinant human NG2 (Origene technologies) was used as standard.

Techniques: Saline, Staining, Marker

Journal: PloS one

Article Title: Pro-inflammatory cytokines reduce the proliferation of NG2 cells and increase shedding of NG2 in vivo and in vitro.

doi: 10.1371/journal.pone.0109387

Figure Lengend Snippet: Figure 4. Proliferation and supernatant sNG2 levels of cultured human NG2 cells after exposure cytokines. The left bar graph shown the significantly increased sNG2 levels found in cell culture supernatant after 24 h stimulation with interleukin (IL)-1b (0.3 ng/ml), IL-6 (1.5 ng/ml) and IL-4 (150 pg/ml). The sNG2 levels were not altered in response to 20 ng/ml IL-8. The right bar graph demonstrates the significantly decreased EdU + cell area to DAPI cell area ratio after exposure to IL-1b and IL-6, but not after IL-8 or IL-4. * indicates a significant difference at p,0.05 level, ** indicates a significant difference at p,0.01 level. doi:10.1371/journal.pone.0109387.g004

Article Snippet: Recombinant rat NG2 (kind gift from Dr William Stallcup) or recombinant human NG2 (Origene technologies) was used as standard.

Techniques: Cell Culture