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Comparative concentration, modal size, and miRNA profiling of CD31<sup>+</sup>...
Published: 23 October 2020
Figure 2 Comparative concentration, modal size, and miRNA profiling of CD31+ EVs from control subjects and patients with T2DM. NTA measurement of the concentration (A) and modal size (B) of CD31+ EVs isolated from healthy control subjects and patients with T2DM (n = 4). C: Comparative cytofluorimetric detection of CD31, CD9, CD63, and CD81 of CD31+ EVs isolated from control subjects and patients with T2DM (n = 4). D: Heat map showing miRNA profiling in CD31+ EVs from control subjects and patients with T2DM (n = 5 vs. 5, pooled samples). *P < 0.05, Student t test. APC, allophycocyanin; a.u., arbitrary units. Figure 2. Comparative concentration, modal size, and miRNA profiling of CD31+ EVs from control subjects and patients with T2DM. NTA measurement of the concentration (A) and modal size (B) of CD31+ EVs isolated from healthy control subjects and patients with T2DM (n = 4). C: Comparative cytofluorimetric detection of CD31, CD9, CD63, and CD81 of CD31+ EVs isolated from control subjects and patients with T2DM (n = 4). D: Heat map showing miRNA profiling in CD31+ EVs from control subjects and patients with T2DM (n = 5 vs. 5, pooled samples). *P < 0.05, Student t test. APC, allophycocyanin; a.u., arbitrary units. More
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Umbilical cord–derived cells exhibit MSC markers. Undifferentiated cells we...
Published: 02 December 2015
Figure 1 Umbilical cord–derived cells exhibit MSC markers. Undifferentiated cells were pooled (more than five subjects) and stained for MSC markers (CD73, CD105, and CD90) and hematopoietic and lymphocyte markers (CD34, CD45, and CD19). Representative plots are shown for MSC expression of CD73 (A), CD105 (B), and CD90 (C), gated as described in research design and methods. Corresponding histograms show gated cells for IgG isotype controls (white) and the marker of interest (gray). The MFI data summary (D) indicates that cells are positive for MSC markers and negative for hematopoietic and lymphocyte markers. APC, allophycocyanin; FITC, fluorescein isothiocyanate; SS, side scatter. Figure 1. Umbilical cord–derived cells exhibit MSC markers. Undifferentiated cells were pooled (more than five subjects) and stained for MSC markers (CD73, CD105, and CD90) and hematopoietic and lymphocyte markers (CD34, CD45, and CD19). Representative plots are shown for MSC expression of CD73 (A), CD105 (B), and CD90 (C), gated as described in research design and methods. Corresponding histograms show gated cells for IgG isotype controls (white) and the marker of interest (gray). The MFI data summary (D) indicates that cells are positive for MSC markers and negative for hematopoietic and lymphocyte markers. APC, allophycocyanin; FITC, fluorescein isothiocyanate; SS, side scatter. More
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Measurements of VEGF and SDF-1 mRNA in circulating SPCs. <em>A</em>...
Published: 20 October 2015
Figure 5 Measurements of VEGF and SDF-1 mRNA in circulating SPCs. A: Gating approach and typical quantification of mRNA. Top left: Laser light forward (FSC-H) and side scatter (SSC-H) with the lymphocyte population circled. Top right: Lymphocyte population and the number of cells identified as CD34+/CD45-dim based on FMO analysis (in the square, 1.91 cells/µL were detected). Bottom two histograms: Fluorescence values (abscissa) and number of events (ordinate) for SDF (left) and VEGF (right) mRNA along with the FMO control. APC, allophycocyanin. B: Relationships between mRNA for SDF-1 and VEGF with HIF ratio. Data show results for nine cell samples with HIF ratio on the abscissa, percent CD34+/CD45-dim cells exhibiting mRNA-specific fluorescence above the FMO baseline as closed circles on the left ordinate axis, and median mRNA fluorescence as open circles on the right ordinate axis. There are no statistically significant associations between mRNA types and HIF ratio. C: Relationship between percent of cells positive (above the FMO baseline) for SDF-1 mRNA and VEGF mRNA. Figure 5. Measurements of VEGF and SDF-1 mRNA in circulating SPCs. A: Gating approach and typical quantification of mRNA. Top left: Laser light forward (FSC-H) and side scatter (SSC-H) with the lymphocyte population circled. Top right: Lymphocyte population and the number of cells identified as CD34+/CD45-dim based on FMO analysis (in the square, 1.91 cells/µL were detected). Bottom two histograms: Fluorescence values (abscissa) and number of events (ordinate) for SDF (left) and VEGF (right) mRNA along with the FMO control. APC, allophycocyanin. B: Relationships between mRNA for SDF-1 and VEGF with HIF ratio. Data show results for nine cell samples with HIF ratio on the abscissa, percent CD34+/CD45-dim cells exhibiting mRNA-specific fluorescence above the FMO baseline as closed circles on the left ordinate axis, and median mRNA fluorescence as open circles on the right ordinate axis. There are no statistically significant associations between mRNA types and HIF ratio. C: Relationship between percent of cells positive (above the FMO baseline) for SDF-1 mRNA and VEGF mRNA. More
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Measurements of VEGF and SDF-1 mRNA in circulating SPCs. <em>A</em>...
Published: 20 October 2015
Figure 5 Measurements of VEGF and SDF-1 mRNA in circulating SPCs. A: Gating approach and typical quantification of mRNA. Top left: Laser light forward (FSC-H) and side scatter (SSC-H) with the lymphocyte population circled. Top right: Lymphocyte population and the number of cells identified as CD34+/CD45-dim based on FMO analysis (in the square, 1.91 cells/µL were detected). Bottom two histograms: Fluorescence values (abscissa) and number of events (ordinate) for SDF (left) and VEGF (right) mRNA along with the FMO control. APC, allophycocyanin. B: Relationships between mRNA for SDF-1 and VEGF with HIF ratio. Data show results for nine cell samples with HIF ratio on the abscissa, percent CD34+/CD45-dim cells exhibiting mRNA-specific fluorescence above the FMO baseline as closed circles on the left ordinate axis, and median mRNA fluorescence as open circles on the right ordinate axis. There are no statistically significant associations between mRNA types and HIF ratio. C: Relationship between percent of cells positive (above the FMO baseline) for SDF-1 mRNA and VEGF mRNA. Figure 5. Measurements of VEGF and SDF-1 mRNA in circulating SPCs. A: Gating approach and typical quantification of mRNA. Top left: Laser light forward (FSC-H) and side scatter (SSC-H) with the lymphocyte population circled. Top right: Lymphocyte population and the number of cells identified as CD34+/CD45-dim based on FMO analysis (in the square, 1.91 cells/µL were detected). Bottom two histograms: Fluorescence values (abscissa) and number of events (ordinate) for SDF (left) and VEGF (right) mRNA along with the FMO control. APC, allophycocyanin. B: Relationships between mRNA for SDF-1 and VEGF with HIF ratio. Data show results for nine cell samples with HIF ratio on the abscissa, percent CD34+/CD45-dim cells exhibiting mRNA-specific fluorescence above the FMO baseline as closed circles on the left ordinate axis, and median mRNA fluorescence as open circles on the right ordinate axis. There are no statistically significant associations between mRNA types and HIF ratio. C: Relationship between percent of cells positive (above the FMO baseline) for SDF-1 mRNA and VEGF mRNA. More
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Gating strategy and representative combinatorial HLA-A3 MMr staining. <ital
Published: 14 September 2020
Figure 1 Gating strategy and representative combinatorial HLA-A3 MMr staining. A: Frozen-thawed PBMCs from donor with T1D D204D were magnetically depleted of CD8 cells before staining, acquisition, and analysis, as described ( 3 ). Cells were sequentially gated on small lymphocytes (tight gate; a loose gate was also applied for comparison in the analysis of PBMCs from donors with T1D and healthy donors; see Fig. 3C and D ), singlets, live cells (Live/Dead [L/D] Aqua), CD3+CD8+ T cells, and total PE+, PE-CF594+, APC+, BV650+, BV711+, and BV786+ MMr+ T cells. Using Boolean operators, these latter gates allowed for selectively visualizing each double-MMr+ population by including only those events positive for the corresponding fluorochrome pair. For example, SCG3166-174 MMr+ cells (PE+PE-CF594+) were visualized by gating on events that were PE+PE-CF594+ and APCBV650BV711BV786. B: The final readout obtained is shown for the 15 peptides analyzed in the validation round on PBMCs from donors with T1D and healthy donors (see Fig. 3 ). Each dot plot displays a color-coded overlay of individual double-MMr+ subsets and of the MMr population (light gray) to visualize the separation of each epitope-reactive CD8+ T-cell fraction relative to the others. The small dot plots on the right of each panel depict CD45RA (x-axis) and CCR7 (y-axis) expression in the corresponding MMr+ fraction. Numbers in each panel indicate the MMr+CD8+ T-cell frequency out of total CD8+ T cells and the percent antigen-experienced fraction among MMr+ cells (i.e., excluding CD45RA+CCR7+ events). APC, allophycocyanin; FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W, forward scatter width; NA, not available; PE, phycoerythrin; SSC-A, side scatter area. Figure 1. Gating strategy and representative combinatorial HLA-A3 MMr staining. A: Frozen-thawed PBMCs from donor with T1D D204D were magnetically depleted of CD8− cells before staining, acquisition, and analysis, as described (3). Cells were sequentially gated on small lymphocytes (tight gate; a loose gate was also applied for comparison in the analysis of PBMCs from donors with T1D and healthy donors; see Fig. 3C and D), singlets, live cells (Live/Dead [L/D] Aqua−), CD3+CD8+ T cells, and total PE+, PE-CF594+, APC+, BV650+, BV711+, and BV786+ MMr+ T cells. Using Boolean operators, these latter gates allowed for selectively visualizing each double-MMr+ population by including only those events positive for the corresponding fluorochrome pair. For example, SCG3166-174 MMr+ cells (PE+PE-CF594+) were visualized by gating on events that were PE+PE-CF594+ and APC−BV650−BV711−BV786−. B: The final readout obtained is shown for the 15 peptides analyzed in the validation round on PBMCs from donors with T1D and healthy donors (see Fig. 3). Each dot plot displays a color-coded overlay of individual double-MMr+ subsets and of the MMr− population (light gray) to visualize the separation of each epitope-reactive CD8+ T-cell fraction relative to the others. The small dot plots on the right of each panel depict CD45RA (x-axis) and CCR7 (y-axis) expression in the corresponding MMr+ fraction. Numbers in each panel indicate the MMr+CD8+ T-cell frequency out of total CD8+ T cells and the percent antigen-experienced fraction among MMr+ cells (i.e., excluding CD45RA+CCR7+ events). APC, allophycocyanin; FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W, forward scatter width; NA, not available; PE, phycoerythrin; SSC-A, side scatter area. More
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The influence of obesity on maturation of DC in AT and liver. Isolated mono...
Published: 17 August 2012
FIG. 6. The influence of obesity on maturation of DC in AT and liver. Isolated mononuclear cells from liver and SVC from AT were stained for markers to define subsets of CD11c+ as well as CD86+ cells and analyzed by flow cytometry (n = minimum of 6 animals per group). Results are presented as means ± SE, and significant differences are indicated (*P < 0.05). A: Number of CD11c+CD86+ cells isolated from AT. B: MFI for CD86 on CD11c+CD86+ cells from AT. C: Number of CD11c+CD86+ cells isolated from liver. D: MFI for CD86 on liver CD11c+CD86+ cells. E: Representative flow cytometry plots of CD86+ cells in AT, corresponding to A and B. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11bCD11c+CD86+ cells. F: Representative flow cytometry plots of CD86+ cells in liver, corresponding to C and D. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11bCD11c+CD86+ cells. FACS, fluorescence-activated cell sorter; APC, allophycocyanin. FIG. 6. The influence of obesity on maturation of DC in AT and liver. Isolated mononuclear cells from liver and SVC from AT were stained for markers to define subsets of CD11c+ as well as CD86+ cells and analyzed by flow cytometry (n = minimum of 6 animals per group). Results are presented as means ± SE, and significant differences are indicated (*P < 0.05). A: Number of CD11c+CD86+ cells isolated from AT. B: MFI for CD86 on CD11c+CD86+ cells from AT. C: Number of CD11c+CD86+ cells isolated from liver. D: MFI for CD86 on liver CD11c+CD86+ cells. E: Representative flow cytometry plots of CD86+ cells in AT, corresponding to A and B. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11b−CD11c+CD86+ cells. F: Representative flow cytometry plots of CD86+ cells in liver, corresponding to C and D. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11b−CD11c+CD86+ cells. FACS, fluorescence-activated cell sorter; APC, allophycocyanin. More
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The influence of obesity on maturation of DC in AT and liver. Isolated mono...
Published: 17 August 2012
FIG. 6. The influence of obesity on maturation of DC in AT and liver. Isolated mononuclear cells from liver and SVC from AT were stained for markers to define subsets of CD11c+ as well as CD86+ cells and analyzed by flow cytometry (n = minimum of 6 animals per group). Results are presented as means ± SE, and significant differences are indicated (*P < 0.05). A: Number of CD11c+CD86+ cells isolated from AT. B: MFI for CD86 on CD11c+CD86+ cells from AT. C: Number of CD11c+CD86+ cells isolated from liver. D: MFI for CD86 on liver CD11c+CD86+ cells. E: Representative flow cytometry plots of CD86+ cells in AT, corresponding to A and B. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11bCD11c+CD86+ cells. F: Representative flow cytometry plots of CD86+ cells in liver, corresponding to C and D. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11bCD11c+CD86+ cells. FACS, fluorescence-activated cell sorter; APC, allophycocyanin. FIG. 6. The influence of obesity on maturation of DC in AT and liver. Isolated mononuclear cells from liver and SVC from AT were stained for markers to define subsets of CD11c+ as well as CD86+ cells and analyzed by flow cytometry (n = minimum of 6 animals per group). Results are presented as means ± SE, and significant differences are indicated (*P < 0.05). A: Number of CD11c+CD86+ cells isolated from AT. B: MFI for CD86 on CD11c+CD86+ cells from AT. C: Number of CD11c+CD86+ cells isolated from liver. D: MFI for CD86 on liver CD11c+CD86+ cells. E: Representative flow cytometry plots of CD86+ cells in AT, corresponding to A and B. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11b−CD11c+CD86+ cells. F: Representative flow cytometry plots of CD86+ cells in liver, corresponding to C and D. Gate 1a represents CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11b−CD11c+CD86+ cells. FACS, fluorescence-activated cell sorter; APC, allophycocyanin. More
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The influence of obesity on CD11b<sup>+</sup>CD11c<sup>+</sup>F4/80<sup>+</sup>...
Published: 17 August 2012
FIG. 3. The influence of obesity on CD11b+CD11c+F4/80+ (triple+) and CD11b+CD11cF4/80+ (double+) cells in AT and liver. Mononuclear cells from liver and stromal vascular cells from AT were isolated from lean and obese mice, stained for CD11b, CD11c, and F4/80 markers, and analyzed by flow cytometry. Data are presented as mean ± SE (minimum number of 6 animals/group analyzed individually). Significant differences are indicated (*P < 0.05). A: Proportion of triple+ cells (CD11b+CD11c+F4/80+) in AT and liver. B: Proportion of double+ cells (CD11b+CD11cF4/80+) in AT and liver. C: Proportion of CD11b+ and CD11b+CD11c cells in AT. D: Proportion of CD11b+ and CD11b+CD11c cells in liver. MC, mononuclear cells. E: Representative flow cytometry plots of triple+ cells in AT and liver, corresponding to A. F: Representative flow cytometry plots of double+ cells in AT and liver, corresponding to B. G: Representative flow cytometry plots of flow cytometry analysis of CD11b+ (gates 3 and 4) and CD11b+CD11c cells (gate 3 only) in AT, corresponding to C. H: Representative flow cytometry plots of CD11b+ (gates 3 and 4) and CD11b+CD11c cells (gate 3 only) in liver, corresponding to D. FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. FIG. 3. The influence of obesity on CD11b+CD11c+F4/80+ (triple+) and CD11b+CD11c−F4/80+ (double+) cells in AT and liver. Mononuclear cells from liver and stromal vascular cells from AT were isolated from lean and obese mice, stained for CD11b, CD11c, and F4/80 markers, and analyzed by flow cytometry. Data are presented as mean ± SE (minimum number of 6 animals/group analyzed individually). Significant differences are indicated (*P < 0.05). A: Proportion of triple+ cells (CD11b+CD11c+F4/80+) in AT and liver. B: Proportion of double+ cells (CD11b+CD11c−F4/80+) in AT and liver. C: Proportion of CD11b+ and CD11b+CD11c− cells in AT. D: Proportion of CD11b+ and CD11b+CD11c− cells in liver. MC, mononuclear cells. E: Representative flow cytometry plots of triple+ cells in AT and liver, corresponding to A. F: Representative flow cytometry plots of double+ cells in AT and liver, corresponding to B. G: Representative flow cytometry plots of flow cytometry analysis of CD11b+ (gates 3 and 4) and CD11b+CD11c− cells (gate 3 only) in AT, corresponding to C. H: Representative flow cytometry plots of CD11b+ (gates 3 and 4) and CD11b+CD11c− cells (gate 3 only) in liver, corresponding to D. FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. More
Images
The influence of obesity on CD11b<sup>+</sup>CD11c<sup>+</sup>F4/80<sup>+</sup>...
Published: 17 August 2012
FIG. 3. The influence of obesity on CD11b+CD11c+F4/80+ (triple+) and CD11b+CD11cF4/80+ (double+) cells in AT and liver. Mononuclear cells from liver and stromal vascular cells from AT were isolated from lean and obese mice, stained for CD11b, CD11c, and F4/80 markers, and analyzed by flow cytometry. Data are presented as mean ± SE (minimum number of 6 animals/group analyzed individually). Significant differences are indicated (*P < 0.05). A: Proportion of triple+ cells (CD11b+CD11c+F4/80+) in AT and liver. B: Proportion of double+ cells (CD11b+CD11cF4/80+) in AT and liver. C: Proportion of CD11b+ and CD11b+CD11c cells in AT. D: Proportion of CD11b+ and CD11b+CD11c cells in liver. MC, mononuclear cells. E: Representative flow cytometry plots of triple+ cells in AT and liver, corresponding to A. F: Representative flow cytometry plots of double+ cells in AT and liver, corresponding to B. G: Representative flow cytometry plots of flow cytometry analysis of CD11b+ (gates 3 and 4) and CD11b+CD11c cells (gate 3 only) in AT, corresponding to C. H: Representative flow cytometry plots of CD11b+ (gates 3 and 4) and CD11b+CD11c cells (gate 3 only) in liver, corresponding to D. FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. FIG. 3. The influence of obesity on CD11b+CD11c+F4/80+ (triple+) and CD11b+CD11c−F4/80+ (double+) cells in AT and liver. Mononuclear cells from liver and stromal vascular cells from AT were isolated from lean and obese mice, stained for CD11b, CD11c, and F4/80 markers, and analyzed by flow cytometry. Data are presented as mean ± SE (minimum number of 6 animals/group analyzed individually). Significant differences are indicated (*P < 0.05). A: Proportion of triple+ cells (CD11b+CD11c+F4/80+) in AT and liver. B: Proportion of double+ cells (CD11b+CD11c−F4/80+) in AT and liver. C: Proportion of CD11b+ and CD11b+CD11c− cells in AT. D: Proportion of CD11b+ and CD11b+CD11c− cells in liver. MC, mononuclear cells. E: Representative flow cytometry plots of triple+ cells in AT and liver, corresponding to A. F: Representative flow cytometry plots of double+ cells in AT and liver, corresponding to B. G: Representative flow cytometry plots of flow cytometry analysis of CD11b+ (gates 3 and 4) and CD11b+CD11c− cells (gate 3 only) in AT, corresponding to C. H: Representative flow cytometry plots of CD11b+ (gates 3 and 4) and CD11b+CD11c− cells (gate 3 only) in liver, corresponding to D. FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. More
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Identification of CD4<sup>+</sup> T-cell frequencies directed against nativ...
Published: 11 October 2018
Figure 4 Identification of CD4+ T-cell frequencies directed against native and citGRP78 peptides in healthy control (HC) subjects and patients with type 1 diabetes (T1D) analyzed by ex vivo Tmr assays. AH: In HC subjects and patients with T1D, respectively, representative FACS plots depict CD4+ T cells in the enriched fraction directed against the native GRP78 peptide 498–512R (A and B), against the citGRP78 peptide 498–512X (C and D), against the native GRP78 peptide 195–209R (E and F), and against the citGRP78 peptide 195–209X (G and H). IL: Total CD4+ T-cell frequencies in the enriched fraction directed against 498–512R (I), 498–512X (J), 195–209R (K), and 195–209X (L) in HC subjects (n = 8; ■) and in patients with T1D (n = 15) subdivided by new-onset T1D (<1 year; n = 4; △) and long-standing T1D (≥1 year; n = 11; ●). CD4+ T-cell frequencies >5 Tmr+ cells/106 (dashed line) are considered positive (indicated in red). MP: Total CD4+ T-cell frequencies in the enriched fraction comparing the native and citrullinated forms of peptide 498–512 in HC subjects (M) and patients in T1D (N) and of peptide 195–209 in HC subjects (O) and patients with T1D (P). Data in IL are mean ± SD and were analyzed by Mann-Whitney U test. *P < 0.05. MP data were analyzed using the Wilcoxon rank sum test. APC, allophycocyanin. Figure 4. Identification of CD4+ T-cell frequencies directed against native and citGRP78 peptides in healthy control (HC) subjects and patients with type 1 diabetes (T1D) analyzed by ex vivo Tmr assays. A–H: In HC subjects and patients with T1D, respectively, representative FACS plots depict CD4+ T cells in the enriched fraction directed against the native GRP78 peptide 498–512R (A and B), against the citGRP78 peptide 498–512X (C and D), against the native GRP78 peptide 195–209R (E and F), and against the citGRP78 peptide 195–209X (G and H). I–L: Total CD4+ T-cell frequencies in the enriched fraction directed against 498–512R (I), 498–512X (J), 195–209R (K), and 195–209X (L) in HC subjects (n = 8; ■) and in patients with T1D (n = 15) subdivided by new-onset T1D (<1 year; n = 4; △) and long-standing T1D (≥1 year; n = 11; ●). CD4+ T-cell frequencies >5 Tmr+ cells/106 (dashed line) are considered positive (indicated in red). M–P: Total CD4+ T-cell frequencies in the enriched fraction comparing the native and citrullinated forms of peptide 498–512 in HC subjects (M) and patients in T1D (N) and of peptide 195–209 in HC subjects (O) and patients with T1D (P). Data in I–L are mean ± SD and were analyzed by Mann-Whitney U test. *P < 0.05. M–P data were analyzed using the Wilcoxon rank sum test. APC, allophycocyanin. More
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Expression of cytotoxicity-related proteins in CD4<sup>+</sup> T cells duri...
Published: 18 January 2022
Figure 4 Expression of cytotoxicity-related proteins in CD4+ T cells during progression to clinical T1D. A: Box plots showing the frequency (Frq.) of CD4+/CXC3CR1+, CD4+/GPR56+, CD4+/GZMB+, and CD4+/perforin+ measured by flow cytometry in progressors (n = 5) and nonprogressors (n = 5) over the period of observation. The x-axis shows time intervals measured in years from time 0 (clinical T1D in progressors). Shown are median (central horizontal line), interquartile range (boxes), values of the upper and lower quartiles (whiskers), and outliers beyond 1.5 interquartile range (circles). Statistical comparisons were performed using linear mixed models that included the progressor status * time point interaction term and were adjusted for age, batch, and donor identifier as a random factor. B: Flow cytometry data for CD4 vs. CX3CR1, CD4 vs. GPR56, CD4 vs. GZMB, and CD4 vs. perforin (after gating on CD3+ and CD4+ T cells) from a representative progressor and nonprogressor. APC, allophycocyanin; PE, phycoerythrin. More
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BM-MSCs from patients with T2D have an increased intrinsic potential to dif...
Published: 27 April 2018
Figure 2 BM-MSCs from patients with T2D have an increased intrinsic potential to differentiate into the adipocyte lineage. A: The bar graph shows basal mRNA expression levels of adipogenesis regulators in BM-MSCs from subjects without diabetes (ND) and subjects with T2D. Values are means with respective SE bars. B: Flow cytometry analysis of BM-MSCs consisted of gating live single cells, selecting for dual-positive CD105-CD73 MSCs fluorescence minus one (FMO; Bi), and finally examining the frequency of cell fractions positive for PDGFRβ (Bii) and PREF-1 (Biii). Scatter plots showing the quantification of CD73posCD105pos (Biv), CD73posCD105posPDGFRβpos (Bv), and CD73posCD105posPREF-1pos (Bvi) cell populations. C: BM-MSC differentiation in ADs. ADs are stained with ORO-positive cells (arrows; Ci) and quantified by morphometry of microscopic images, with values in the ND and T2D groups illustrated by the scatter plot (Cii). Bar graphs show the results from qPCR analysis of PPARγ (Ciii), ADIPOQ (Civ), and FABP4 (Cv) mRNA expression in BM-MSCs at different times of the adipogenesis induction assay. Values are means with SE bars. *P < 0.05; **P < 0.01; ***P < 0.001 vs. subjects without diabetes; +P < 0.05; +++P < 0.001 vs. time 0 of the adipogenic assay. D: BM-MSC differentiation in osteoblasts, detected by staining with alkaline phosphatase. Representative images with positive cells indicated by arrows (Di) and scatter plot showing means with SE bars (Dii). No difference was detected in this comparison. Student t test with unequal distribution was used in all of the comparisons. N = 4 biological replicates/group. Scale bars, 250 μm. APC, allophycocyanin; FITC, fluorescein isothiocyanate. Figure 2. BM-MSCs from patients with T2D have an increased intrinsic potential to differentiate into the adipocyte lineage. A: The bar graph shows basal mRNA expression levels of adipogenesis regulators in BM-MSCs from subjects without diabetes (ND) and subjects with T2D. Values are means with respective SE bars. B: Flow cytometry analysis of BM-MSCs consisted of gating live single cells, selecting for dual-positive CD105-CD73 MSCs fluorescence minus one (FMO; Bi), and finally examining the frequency of cell fractions positive for PDGFRβ (Bii) and PREF-1 (Biii). Scatter plots showing the quantification of CD73posCD105pos (Biv), CD73posCD105posPDGFRβpos (Bv), and CD73posCD105posPREF-1pos (Bvi) cell populations. C: BM-MSC differentiation in ADs. ADs are stained with ORO-positive cells (arrows; Ci) and quantified by morphometry of microscopic images, with values in the ND and T2D groups illustrated by the scatter plot (Cii). Bar graphs show the results from qPCR analysis of PPARγ (Ciii), ADIPOQ (Civ), and FABP4 (Cv) mRNA expression in BM-MSCs at different times of the adipogenesis induction assay. Values are means with SE bars. *P < 0.05; **P < 0.01; ***P < 0.001 vs. subjects without diabetes; +P < 0.05; +++P < 0.001 vs. time 0 of the adipogenic assay. D: BM-MSC differentiation in osteoblasts, detected by staining with alkaline phosphatase. Representative images with positive cells indicated by arrows (Di) and scatter plot showing means with SE bars (Dii). No difference was detected in this comparison. Student t test with unequal distribution was used in all of the comparisons. N = 4 biological replicates/group. Scale bars, 250 μm. APC, allophycocyanin; FITC, fluorescein isothiocyanate. More
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Isolation and characterization of CD31<sup>+</sup> EVs. <em>A</em>:...
Published: 23 October 2020
Figure 1 Isolation and characterization of CD31+ EVs. A: Schematic representation of the isolation method. B: NTA of one representative sample of isolated CD31+ EVs, along with the observed mean size and number (from 1 mL pooled control plasma). C: Representative TEM image of EVs isolated with CD31 beads along with the relative magnification. D: Western blot showing the expression of CD31, Alix, TSG101, CD63, apoB100, and apoA1 in CD31+ EVs and UC-collected EVs isolated from the same amount of plasma, along with the relative densitometric analysis. Whole plasma was run as positive control for apoA1 and apoB100. E: Ratio between the expression of CD31 and CD9, CD63, or CD81 in CD31+ EVs and that in EVs isolated through UC, as measured with a specific kit allowing cytofluorimetric detection (n = 6 from pooled plasma split for performance of comparative isolation starting from the same volume). F: Comparative cytofluorimetric detection of CD49e, CD9, CD63, CD62P, CD81, CD41b, CD42a, CD29, and CD69 in EVs isolated with no beads, scramble IgG beads, and CD31 beads (n = 3, from equal amount of control plasma samples). G: Concentration of collected CD31+ EVs vs. the CD31-depleted fraction of EVs subjected to UC, measured with standard NTA (n = 3). H: RT-PCR dosage of miR-126-3p, miR-146a-5p, miR-155, and miR-21-5p in whole plasma vs. total EVs isolated with UC vs. CD31+ EVs, with division of the same control samples in different aliquots (same volume, 100 μL) for comparison of the relative abundance in the various compartments (n = 8). Errors bar are ±SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student t test for panels D, E, and G and one-way ANOVA for panels F and H. #P < 0.05 vs. UC for panel H. APC, allophycocyanin; a.u., arbitrary units. Figure 1. Isolation and characterization of CD31+ EVs. A: Schematic representation of the isolation method. B: NTA of one representative sample of isolated CD31+ EVs, along with the observed mean size and number (from 1 mL pooled control plasma). C: Representative TEM image of EVs isolated with CD31 beads along with the relative magnification. D: Western blot showing the expression of CD31, Alix, TSG101, CD63, apoB100, and apoA1 in CD31+ EVs and UC-collected EVs isolated from the same amount of plasma, along with the relative densitometric analysis. Whole plasma was run as positive control for apoA1 and apoB100. E: Ratio between the expression of CD31 and CD9, CD63, or CD81 in CD31+ EVs and that in EVs isolated through UC, as measured with a specific kit allowing cytofluorimetric detection (n = 6 from pooled plasma split for performance of comparative isolation starting from the same volume). F: Comparative cytofluorimetric detection of CD49e, CD9, CD63, CD62P, CD81, CD41b, CD42a, CD29, and CD69 in EVs isolated with no beads, scramble IgG beads, and CD31 beads (n = 3, from equal amount of control plasma samples). G: Concentration of collected CD31+ EVs vs. the CD31-depleted fraction of EVs subjected to UC, measured with standard NTA (n = 3). H: RT-PCR dosage of miR-126-3p, miR-146a-5p, miR-155, and miR-21-5p in whole plasma vs. total EVs isolated with UC vs. CD31+ EVs, with division of the same control samples in different aliquots (same volume, 100 μL) for comparison of the relative abundance in the various compartments (n = 8). Errors bar are ±SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student t test for panels D, E, and G and one-way ANOVA for panels F and H. #P < 0.05 vs. UC for panel H. APC, allophycocyanin; a.u., arbitrary units. More
Images
Maternal DEX administration inhibits <em>Ppargc1a</em> expression a...
Published: 14 May 2020
Figure 4 Maternal DEX administration inhibits Ppargc1a expression and mitochondrial biogenesis of brown progenitors in offspring BAT at 4 months of age. A and B: Flow cytometry analyses (FACS) in sorting and measuring the population of brown progenitors in offspring BAT using brown progenitor markers Lin CD45/PDGFRa+ (also named as CD140). Positive population of brown progenitors shown in black ovals (A) and quantified in B (n = 6 per group). CF: Immunoblotting in measuring protein content of PGC-1a (D), CYTO-C (E), and VDAC (F) in sorted brown progenitors in offspring BAT (n = 6 per group). β-Tubulin was used as a loading control. G: mtDNA copy of brown progenitors isolated in offspring BAT. Amplification of mitochondrial genes was standardized to 18S rRNA and GAPDH. n = 6 per group. H and I: Immunostaining (H) and fluorophore intensity (I) of mitochondria (Mito) in sorted brown progenitors using MitoSpy (green). Scale bars, 100 μm. JL: Cell proliferation of brown progenitors was measured using MTT and BrdU assays. Progenitor cells were treated with BrdU for 24 h followed by BrdU and DAPI immunostaining (K); scale bars, 200 μm. Proliferative cells were displayed as BrdU+ cells (L). M and N: Brown progenitors isolated from offspring BAT were induced into brown adipocytes using standard brown adipogenic differentiation protocol in vitro followed by lipid staining (M) and quantification (N) using oil red O (n = 4 per group). Data are means ± SEM, and each dot represents one replicate (litter). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t test with two-tailed distribution was used in analyses. APC, allophycocyanin; A.U., arbitrary units; CON, control; PE, phycoerythrin; Rel., relative. Figure 4. Maternal DEX administration inhibits Ppargc1a expression and mitochondrial biogenesis of brown progenitors in offspring BAT at 4 months of age. A and B: Flow cytometry analyses (FACS) in sorting and measuring the population of brown progenitors in offspring BAT using brown progenitor markers Lin− CD45−/PDGFRa+ (also named as CD140). Positive population of brown progenitors shown in black ovals (A) and quantified in B (n = 6 per group). C–F: Immunoblotting in measuring protein content of PGC-1a (D), CYTO-C (E), and VDAC (F) in sorted brown progenitors in offspring BAT (n = 6 per group). β-Tubulin was used as a loading control. G: mtDNA copy of brown progenitors isolated in offspring BAT. Amplification of mitochondrial genes was standardized to 18S rRNA and GAPDH. n = 6 per group. H and I: Immunostaining (H) and fluorophore intensity (I) of mitochondria (Mito) in sorted brown progenitors using MitoSpy (green). Scale bars, 100 μm. J–L: Cell proliferation of brown progenitors was measured using MTT and BrdU assays. Progenitor cells were treated with BrdU for 24 h followed by BrdU and DAPI immunostaining (K); scale bars, 200 μm. Proliferative cells were displayed as BrdU+ cells (L). M and N: Brown progenitors isolated from offspring BAT were induced into brown adipocytes using standard brown adipogenic differentiation protocol in vitro followed by lipid staining (M) and quantification (N) using oil red O (n = 4 per group). Data are means ± SEM, and each dot represents one replicate (litter). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t test with two-tailed distribution was used in analyses. APC, allophycocyanin; A.U., arbitrary units; CON, control; PE, phycoerythrin; Rel., relative. More
Images
The influence of HFD on CD11c<sup>+</sup> cells. <em>A</em> and ...
Published: 17 August 2012
FIG. 2. The influence of HFD on CD11c+ cells. A and B: After 26 weeks of dietary exposure, mononuclear cells were isolated from spleen (Spl), adipose tissue (AT), liver (Liv), and MLN and stained for specific markers, then analyzed by flow cytometry. A: Number of CD11c+ cells in tissues. B: Proportion of CD11c+ cells among total mononuclear cells isolated from spleen, liver, and MLNs and SVC from AT. C: Fold changes in liver CD11c+ cells in 3-week SCD or HFD or 3-week SCD or HFD followed by the SCD for additional 3 weeks (3 + 3 weeks). D: Representative flow cytometry plots of CD11c+ cells (gate 1) isolated from AT and liver of lean and obese animals. For all experiments above, a minimum of 6 animals per group were individually analyzed (n = 6). Results are presented as means ± SE. Significant differences are indicated (*P < 0.05). E: Representative immunofluorescence of epididymal fat pads obtained from lean and obese animals. After fixation in 2% paraformaldehyde, ∼1 mm3 of tissue was labeled in suspension using rat anti-mouse F4/80 (clone 6F12) and hamster anti-mouse CD11c, both at 1:100 dilution (BD Pharminogen). Goat anti-rat Alexa Fluor 488 (1:500; Invitrogen) and goat anti-hamster Cy3 (1:1,000; Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Confocal stack tissue reconstructions of 50 µm were taken at 5 µm intervals using an Olympus Fluoview 1000 Microscope (Olympus, Center Valley, PA). FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. (A high-quality digital representation of this figure is available in the online issue.) FIG. 2. The influence of HFD on CD11c+ cells. A and B: After 26 weeks of dietary exposure, mononuclear cells were isolated from spleen (Spl), adipose tissue (AT), liver (Liv), and MLN and stained for specific markers, then analyzed by flow cytometry. A: Number of CD11c+ cells in tissues. B: Proportion of CD11c+ cells among total mononuclear cells isolated from spleen, liver, and MLNs and SVC from AT. C: Fold changes in liver CD11c+ cells in 3-week SCD or HFD or 3-week SCD or HFD followed by the SCD for additional 3 weeks (3 + 3 weeks). D: Representative flow cytometry plots of CD11c+ cells (gate 1) isolated from AT and liver of lean and obese animals. For all experiments above, a minimum of 6 animals per group were individually analyzed (n = 6). Results are presented as means ± SE. Significant differences are indicated (*P < 0.05). E: Representative immunofluorescence of epididymal fat pads obtained from lean and obese animals. After fixation in 2% paraformaldehyde, ∼1 mm3 of tissue was labeled in suspension using rat anti-mouse F4/80 (clone 6F12) and hamster anti-mouse CD11c, both at 1:100 dilution (BD Pharminogen). Goat anti-rat Alexa Fluor 488 (1:500; Invitrogen) and goat anti-hamster Cy3 (1:1,000; Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. Confocal stack tissue reconstructions of 50 µm were taken at 5 µm intervals using an Olympus Fluoview 1000 Microscope (Olympus, Center Valley, PA). FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; APC, allophycocyanin. (A high-quality digital representation of this figure is available in the online issue.) More
Images
Maternal DEX inhibits <em>Ppargc1a</em> expression and mitochondria...
Published: 14 May 2020
Figure 6 Maternal DEX inhibits Ppargc1a expression and mitochondrial biogenesis in fetal BAT and brown progenitors. AC: After birth (P0), neonatal BAT and brown progenitors isolated from BAT were used for measurement of Ppargc1a mRNA (A) and protein contents of PGC-1a, GR, CYTO-C, VDAC, and UCP-1 (B and C). mRNA expression was standardized to 18S rRNA, and β-actin was used as a loading control in Western blot. D: TEM imaging displayed mitochondrial density in neonatal BAT (scale bar, ×3,500 for 2 μm, ×6,500 for 1 μm). E: Thermal imaging of the surface temperature in the interscapular region of neonates at P0 (n = 6 per group). Measurements were performed immediately after neonates were separated from the nests in order to avoid heat loss at room temperature. F: Immunostaining and intensity of mitochondria (Mito) in brown progenitors sorted from neonatal BAT. MitoSpy (green) was used as a mitochondrial tracker. G and H: FACS for measuring the population of brown progenitors in neonatal BAT using progenitor markers: Lin CD45/PDGFRa+. Positive population of brown progenitors shown in black ovals (G) and quantified in H (n = 6 per group). I: BAT mass (% body mass) in neonates at P0 and offspring at weaning P21 (n = 6 per group). J and K: Correlation between 5mC abundance and mRNA expression of Ppargc1a in offspring BAT (J) and brown progenitors (K). Statistical analyses were assessed by linear and higher-order nonlinear regressions, respectively, and Bayesian information criterion was used for model selection among a finite set of regressions. With use of random Legendre regression analyses, second-degree polynormal regression model identified a higher prediction likelihood R2. Data are means ± SEM, and each dot represents one replicate (litter). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t test with two-tailed distribution was used in analyses. APC, allophycocyanin; A.U., arbitrary units; CON, control; iBAT, intrascapular BAT; Rel., relative. Figure 6. Maternal DEX inhibits Ppargc1a expression and mitochondrial biogenesis in fetal BAT and brown progenitors. A–C: After birth (P0), neonatal BAT and brown progenitors isolated from BAT were used for measurement of Ppargc1a mRNA (A) and protein contents of PGC-1a, GR, CYTO-C, VDAC, and UCP-1 (B and C). mRNA expression was standardized to 18S rRNA, and β-actin was used as a loading control in Western blot. D: TEM imaging displayed mitochondrial density in neonatal BAT (scale bar, ×3,500 for 2 μm, ×6,500 for 1 μm). E: Thermal imaging of the surface temperature in the interscapular region of neonates at P0 (n = 6 per group). Measurements were performed immediately after neonates were separated from the nests in order to avoid heat loss at room temperature. F: Immunostaining and intensity of mitochondria (Mito) in brown progenitors sorted from neonatal BAT. MitoSpy (green) was used as a mitochondrial tracker. G and H: FACS for measuring the population of brown progenitors in neonatal BAT using progenitor markers: Lin− CD45−/PDGFRa+. Positive population of brown progenitors shown in black ovals (G) and quantified in H (n = 6 per group). I: BAT mass (% body mass) in neonates at P0 and offspring at weaning P21 (n = 6 per group). J and K: Correlation between 5mC abundance and mRNA expression of Ppargc1a in offspring BAT (J) and brown progenitors (K). Statistical analyses were assessed by linear and higher-order nonlinear regressions, respectively, and Bayesian information criterion was used for model selection among a finite set of regressions. With use of random Legendre regression analyses, second-degree polynormal regression model identified a higher prediction likelihood R2. Data are means ± SEM, and each dot represents one replicate (litter). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t test with two-tailed distribution was used in analyses. APC, allophycocyanin; A.U., arbitrary units; CON, control; iBAT, intrascapular BAT; Rel., relative. More
Journal Articles
Journal: Diabetes
Diabetes 2012;61(9):2330–2339
Published: 17 August 2012
... CD11c+CD86+ cells; gate 4a represents CD11b+CD11c+CD86+ cells; and gate 5a represents CD11bCD11c+CD86+ cells. FACS, fluorescence-activated cell sorter; APC, allophycocyanin. FIG. 6. The influence of obesity...
Includes: Supplementary data
Journal Articles
Journal: Diabetes
Diabetes 2020;69(8):1662–1674
Published: 14 May 2020
... < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student t test with two-tailed distribution was used in analyses. APC, allophycocyanin; A.U., arbitrary units; CON, control; PE, phycoerythrin; Rel., relative. Figure 4. Maternal DEX administration...
Journal Articles
Journal: Diabetes
Diabetes 2016;65(3):647–659
Published: 02 December 2015
... and negative for hematopoietic and lymphocyte markers. APC, allophycocyanin; FITC, fluorescein isothiocyanate; SS, side scatter. Figure 1. Umbilical cord–derived cells exhibit MSC markers. Undifferentiated cells were pooled (more than five subjects) and stained for MSC markers (CD73, CD105, and CD90...
Includes: Supplementary data
Images
CD13/APN content is greater in Ob-MSCs. Undifferentiated cells from each su...
Published: 02 December 2015
Figure 3 CD13/APN content is greater in Ob-MSCs. Undifferentiated cells from each subject were stained for CD13/APN. Data shown are for the allophycocyanin (APC) IgG isotype control (A) and representative NW-MSC (B) and Ob-MSC (C) flow plots for CD13 APCs. For A–C, graphs are the light scatter gate (first graph), live/dead gate (second graph), singlet gate (third graph), and APC fluorescence intensity (fourth graph) showing that NW-MSCs and Ob-MSCs have similar size, density, and viability. D: The percent of cells positive for CD13/APN was similar for NW-MSCs and Ob-MSCs (black bars and white bars, respectively), though the CD13/APN APC fluorescence intensity, shown from representative subjects (E) and a data summary (F), is higher in Ob-MSCs. Data are expressed as mean ± SEM. *Significant difference from NW-MSCs (P ≤ 0.05). FS, forward scatter; SS, side scatter. Figure 3. CD13/APN content is greater in Ob-MSCs. Undifferentiated cells from each subject were stained for CD13/APN. Data shown are for the allophycocyanin (APC) IgG isotype control (A) and representative NW-MSC (B) and Ob-MSC (C) flow plots for CD13 APCs. For A–C, graphs are the light scatter gate (first graph), live/dead gate (second graph), singlet gate (third graph), and APC fluorescence intensity (fourth graph) showing that NW-MSCs and Ob-MSCs have similar size, density, and viability. D: The percent of cells positive for CD13/APN was similar for NW-MSCs and Ob-MSCs (black bars and white bars, respectively), though the CD13/APN APC fluorescence intensity, shown from representative subjects (E) and a data summary (F), is higher in Ob-MSCs. Data are expressed as mean ± SEM. *Significant difference from NW-MSCs (P ≤ 0.05). FS, forward scatter; SS, side scatter. More