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fitc-fluorescein-isothiocyanate

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Cpd K effects on the generation of CD4<sup>+</sup> and CD8<sup>+</sup> T ce...
Published: 15 September 2014
Figure 5 Cpd K effects on the generation of CD4+ and CD8+ T cells. Proportion of splenic and lymph node CD4+ and CD8+ T cells were analyzed by flow cytometry at day 10 posttransplantation (n = 3 mice per group). The numbers are for representative data of three independent experiments. FITC, fluorescein isothiocyanate. Figure 5. Cpd K effects on the generation of CD4+ and CD8+ T cells. Proportion of splenic and lymph node CD4+ and CD8+ T cells were analyzed by flow cytometry at day 10 posttransplantation (n = 3 mice per group). The numbers are for representative data of three independent experiments. FITC, fluorescein isothiocyanate. More
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Flow cytometry blood SPC analysis for HIF proteins. After forward (FSC-H) a...
Published: 20 October 2015
Figure 3 Flow cytometry blood SPC analysis for HIF proteins. After forward (FSC-H) and side scatter (SSC-H) interrogation, the P1 zone was selected and cells positive for CD34 and negative for CD45 (defined based on the FMO Control Test [box P3] were analyzed for content of HIF proteins). Histogra... More
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Intracellular localization of AAT in MIN6 cells. <em>A</em>: Fluore...
Published: 01 May 2007
FIG. 4. Intracellular localization of AAT in MIN6 cells. A: Fluorescent-labeled AAT entered MIN6 cells and showed a punctuated pattern in the cytoplasm. To observe this, cells were incubated with labeled AAT for 6 h and washed three times with saline. B: Immunostaining with rabbit anti-hAAT antibody. For this procedure, cells were incubated with AAT for 6 h. C: Immunostaining for AATα and nuclear staining showed that the majority of hAAT was in cytoplasm. FITC, fluorescein isothiocyanate. FIG. 4. Intracellular localization of AAT in MIN6 cells. A: Fluorescent-labeled AAT entered MIN6 cells and showed a punctuated pattern in the cytoplasm. To observe this, cells were incubated with labeled AAT for 6 h and washed three times with saline. B: Immunostaining with rabbit anti-hAAT antibody. For this procedure, cells were incubated with AAT for 6 h. C: Immunostaining for AATα and nuclear staining showed that the majority of hAAT was in cytoplasm. FITC, fluorescein isothiocyanate. More
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The apoptotic effect of TMr-GAD (10 μg/ml, 12 h) is due to activation-induc...
Published: 01 April 2004
FIG. 7. The apoptotic effect of TMr-GAD (10 μg/ml, 12 h) is due to activation-induced cell death. A: Dose-dependent release of sFasL by TMr-GAD–treated BRI4.13 cells, as measured by ELISA. B: Inhibition of TMr-GAD–induced apoptosis by a blocking anti-FasL mAb. C: Potentiation of TMr-GAD–induced apoptosis by a matrix metalloproteinase inhibitor (MMPI) blocking the release of sFasL. D: Concomitant increase in surface FasL expression. FITC, fluorescein isothiocyanate. FIG. 7. The apoptotic effect of TMr-GAD (10 μg/ml, 12 h) is due to activation-induced cell death. A: Dose-dependent release of sFasL by TMr-GAD–treated BRI4.13 cells, as measured by ELISA. B: Inhibition of TMr-GAD–induced apoptosis by a blocking anti-FasL mAb. C: Potentiation of TMr-GAD–induced apoptosis by a matrix metalloproteinase inhibitor (MMPI) blocking the release of sFasL. D: Concomitant increase in surface FasL expression. FITC, fluorescein isothiocyanate. More
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TMr-GAD–induced apoptosis of T-cells. Cells were cultured for 12 h in the p...
Published: 01 April 2004
FIG. 6. TMr-GAD–induced apoptosis of T-cells. Cells were cultured for 12 h in the presence of TMrs at the indicated concentrations. AD: Histograms show the ann-V staining of stimulated cells (solid lines) and the percent increase in apoptotic (ann-V+) cells compared with the basal unstimulated condition (dotted lines) for the BRI4.13 (AD) and BRI4.1 (FH) clones. E: Three-dimensional plots of forward and side scatter distribution among TMr-MBP–and TMr-GAD–treated BRI4.13 and BRI4.1 clones. FITC, fluorescein isothiocyanate. FIG. 6. TMr-GAD–induced apoptosis of T-cells. Cells were cultured for 12 h in the presence of TMrs at the indicated concentrations. A–D: Histograms show the ann-V staining of stimulated cells (solid lines) and the percent increase in apoptotic (ann-V+) cells compared with the basal unstimulated condition (dotted lines) for the BRI4.13 (A–D) and BRI4.1 (F–H) clones. E: Three-dimensional plots of forward and side scatter distribution among TMr-MBP–and TMr-GAD–treated BRI4.13 and BRI4.1 clones. FITC, fluorescein isothiocyanate. More
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NEFA induces NF-κB activation in VSMC. <em>A</em>: a representative...
Published: 03 March 2010
FIG. 6. NEFA induces NF-κB activation in VSMC. A: a representative EMSA is shown. B: Bar graph showing NF-κB activation in VSMCs incubated for 3 h with increasing linoleate (LA) concentrations. C: p65 subunit of NF-κB was detected by indirect immunostaining using FITC: Fluorescein isothiocyanate–labeled secondary antibodies and evaluated by confocal microscopy. Figure shows a representative experiment where rat VSMCs were incubated with or without 90 μmol/l linoleate. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.) FIG. 6. NEFA induces NF-κB activation in VSMC. A: a representative EMSA is shown. B: Bar graph showing NF-κB activation in VSMCs incubated for 3 h with increasing linoleate (LA) concentrations. C: p65 subunit of NF-κB was detected by indirect immunostaining using FITC: Fluorescein isothiocyanate–labeled secondary antibodies and evaluated by confocal microscopy. Figure shows a representative experiment where rat VSMCs were incubated with or without 90 μmol/l linoleate. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.) More
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LTβR-Ig–treated animals had normal splenic cellularity and architecture. Th...
Published: 01 December 2004
FIG. 1. LTβR-Ig–treated animals had normal splenic cellularity and architecture. The cellularity of the spleens from control immunoglobulin and LTβR-Ig mice were not different from one another (A). There was no statistically significant difference in the total number of cells or in the numbers of T-cells, B-cells, dendritic cells, and macrophages. The architecture of the spleens appeared normal in the nodeless mice. The white pulp contained a normal appearing periarteriolar lymphoid sheath (anti–CD3-FITC ([fluorescein isothiocyanate]: green) surrounded by normal appearing B-cells (anti–CD-19-PE: red) (B). FIG. 1. LTβR-Ig–treated animals had normal splenic cellularity and architecture. The cellularity of the spleens from control immunoglobulin and LTβR-Ig mice were not different from one another (A). There was no statistically significant difference in the total number of cells or in the numbers of T-cells, B-cells, dendritic cells, and macrophages. The architecture of the spleens appeared normal in the nodeless mice. The white pulp contained a normal appearing periarteriolar lymphoid sheath (anti–CD3-FITC ([fluorescein isothiocyanate]: green) surrounded by normal appearing B-cells (anti–CD-19-PE: red) (B). 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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Primary duct cells express TF. <em>A</em>: Primary duct cells cultu...
Published: 01 June 2004
FIG. 1. Primary duct cells express TF. A: Primary duct cells cultured in monolayers were dissociated and analyzed by flow cytometry after staining by either anti-TF IgG mAb (thick line) or corresponding control isotype mAb (dotted line). A similar staining was found in four independent experiments. B: Immunohistochemical analysis for TF and CK19 was performed on serial sections of formalin-fixed, paraffin-embedded, human normal pancreatic specimens. Left panel: TF staining of epithelial cells. Right panel: CK19 staining on a consecutive section (original magnification 400×). FITC, fluorescein isothiocyanate. FIG. 1. Primary duct cells express TF. A: Primary duct cells cultured in monolayers were dissociated and analyzed by flow cytometry after staining by either anti-TF IgG mAb (thick line) or corresponding control isotype mAb (dotted line). A similar staining was found in four independent experiments. B: Immunohistochemical analysis for TF and CK19 was performed on serial sections of formalin-fixed, paraffin-embedded, human normal pancreatic specimens. Left panel: TF staining of epithelial cells. Right panel: CK19 staining on a consecutive section (original magnification 400×). FITC, fluorescein isothiocyanate. More
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Isolation, derivation, and characterization of clonal mBMDS cells. <em></em>...
Published: 01 July 2004
FIG. 1. Isolation, derivation, and characterization of clonal mBMDS cells. A: BM cells (2 × 106 cells/ml) from Balb/c mice were plated and cultured for 2–7 days to obtain the adherent mBMDS cells (top). Cloned mBMDS cells were used for the in vitro differentiation protocol by culturing cells in the presence of a 23-mmol/l glucose concentration for various times. Many cell clusters at various stages of cluster formation during the course of induction of cell differentiation were observed, with a representative shown in the bottom panel. HG, high glucose; LG, low glucose. B: A representative phenotype of the mBMDS cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin. FIG. 1. Isolation, derivation, and characterization of clonal mBMDS cells. A: BM cells (2 × 106 cells/ml) from Balb/c mice were plated and cultured for 2–7 days to obtain the adherent mBMDS cells (top). Cloned mBMDS cells were used for the in vitro differentiation protocol by culturing cells in the presence of a 23-mmol/l glucose concentration for various times. Many cell clusters at various stages of cluster formation during the course of induction of cell differentiation were observed, with a representative shown in the bottom panel. HG, high glucose; LG, low glucose. B: A representative phenotype of the mBMDS cells. FITC, fluorescein isothiocyanate; PE, phycoerythrin. More
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Representative flow cytomteric analyses of gated CD4 T cell–naive (CD45R0...
Published: 15 September 2014
Figure 2 Representative flow cytomteric analyses of gated CD4 T cell–naive (CD45R0CD62L+) and CM T cell (CD45R0+CD62L+) subpopulations at different time points during the study in which patients were receiving maintenance therapy (abatacept or placebo) when tested at 3, 6, 12, and 24 months, at which point treatment ceased. Numbers in quadrants are the percentages of each subset. A: Lysed whole-blood staining of a type 1 diabetes patient from the placebo group. There is no notable change in the percentage of CM cells (top right quadrant) or the percentage of naive cells (bottom right quadrant). B: A patient in the abatacept-treated arm of the study, in whom there is marked change in the proportion of circulating CM (reduced) and naive (increased) CD4 T cells, respectively. FITC, fluorescein isothiocyanate. Figure 2. Representative flow cytomteric analyses of gated CD4 T cell–naive (CD45R0−CD62L+) and CM T cell (CD45R0+CD62L+) subpopulations at different time points during the study in which patients were receiving maintenance therapy (abatacept or placebo) when tested at 3, 6, 12, and 24 months, at which point treatment ceased. Numbers in quadrants are the percentages of each subset. A: Lysed whole-blood staining of a type 1 diabetes patient from the placebo group. There is no notable change in the percentage of CM cells (top right quadrant) or the percentage of naive cells (bottom right quadrant). B: A patient in the abatacept-treated arm of the study, in whom there is marked change in the proportion of circulating CM (reduced) and naive (increased) CD4 T cells, respectively. FITC, fluorescein isothiocyanate. More
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3T3-L1 cells treated with C8-CPPC and/or rosiglitazone. <em>A</em> ...
Published: 28 October 2014
Figure 4 3T3-L1 cells treated with C8-CPPC and/or rosiglitazone. A and B: mRNA expression of genes involved in adipocyte differentiation and lipid accumulation at 24 and 48 h after induction. C: Protein expression levels of PPARγ2 and cEBPα. D: Lipid content after 48 and 96 h of differentiation. Values are the mean ± SEM of two separate experiments performed in triplicate. One-way ANOVA was used to analyze the statistical significance between treatments at 24 and 48 h. Significant differences (Duncan test, P < 0.05) are indicated with different letters. E: DhCer, Cer, and hexosylceramide (HexCer) levels after 48 h of differentiation. Values are the mean ± SEM of two separate experiments performed in triplicate. cEBP, CCAAT/enhancer binding protein; d, day; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; Rosi, rosiglitazone. *P < 0.05. Figure 4. 3T3-L1 cells treated with C8-CPPC and/or rosiglitazone. A and B: mRNA expression of genes involved in adipocyte differentiation and lipid accumulation at 24 and 48 h after induction. C: Protein expression levels of PPARγ2 and cEBPα. D: Lipid content after 48 and 96 h of differentiation. Values are the mean ± SEM of two separate experiments performed in triplicate. One-way ANOVA was used to analyze the statistical significance between treatments at 24 and 48 h. Significant differences (Duncan test, P < 0.05) are indicated with different letters. E: DhCer, Cer, and hexosylceramide (HexCer) levels after 48 h of differentiation. Values are the mean ± SEM of two separate experiments performed in triplicate. cEBP, CCAAT/enhancer binding protein; d, day; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; Rosi, rosiglitazone. *P < 0.05. More
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TCF7L2 knockdown in mouse pancreatic islets and INS-1(832/13) β-cells does ...
Published: 23 January 2009
FIG. 2. TCF7L2 knockdown in mouse pancreatic islets and INS-1(832/13) β-cells does not alter glucose-stimulated changes in intracellular free [Ca2+] but alters total cellular ATP and ADP levels. Mouse islets were treated and intracellular adenine nucleotide measured as described in research design and methods (A). Dissociated mouse islet cells (B and C) and INS-1(832/13) β-cells (D and E) were treated with 10 nmol/l fluorescein-labeled siRNA (B and C) or pEGFP-TCF7L2 shRNA (D and E) and incubated with FURA-Red (200 nmol/l; B–E) for [Ca2+]i measurement. Typical traces are shown (C and D). Glucose-induced (3.0 versus 11 mmol/l) [Ca2+]i changes in individual glucose-responsive dissociated mouse islet cells in which TCF7L2 was silenced (B) and the number of INS-1(832/13) cells within the cell population that responded to (20 mmol/l) glucose (E) are shown. Data are means ± SEM, n = 3, unless otherwise stated in the research design and methods. AUC, area under the curve; FITC, fluorescein isothiocyanate. FIG. 2. TCF7L2 knockdown in mouse pancreatic islets and INS-1(832/13) β-cells does not alter glucose-stimulated changes in intracellular free [Ca2+] but alters total cellular ATP and ADP levels. Mouse islets were treated and intracellular adenine nucleotide measured as described in research design and methods (A). Dissociated mouse islet cells (B and C) and INS-1(832/13) β-cells (D and E) were treated with 10 nmol/l fluorescein-labeled siRNA (B and C) or pEGFP-TCF7L2 shRNA (D and E) and incubated with FURA-Red (200 nmol/l; B–E) for [Ca2+]i measurement. Typical traces are shown (C and D). Glucose-induced (3.0 versus 11 mmol/l) [Ca2+]i changes in individual glucose-responsive dissociated mouse islet cells in which TCF7L2 was silenced (B) and the number of INS-1(832/13) cells within the cell population that responded to (20 mmol/l) glucose (E) are shown. Data are means ± SEM, n = 3, unless otherwise stated in the research design and methods. AUC, area under the curve; FITC, fluorescein isothiocyanate. More
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Representative examples of pSTAT5a staining, the gating used to define CD4...
Published: 29 July 2015
Figure 2 Representative examples of pSTAT5a staining, the gating used to define CD4+ T-cell populations, and IL-2 dose response. Examples of pSTAT5a staining in CD4+ T cells upon stimulation with 0.1 (A), 0.25 (B) and 10 IU/mL (C) of IL-2 for 30 min, using barcoded cryopreserved PBMCs stained with CD4, CD25, CD45RA, and pSTAT5a. D: Tregs were identified using two different gating strategies: based on a high level of CD25 staining and reduced CD4 staining (CD4loCD25+ Tregs) (i) and gated on the top 2% of CD25-staining CD4+ cells (CD25hi Tregs) (ii). Tconv were identified by low/intermediate levels of CD25 staining. FITC, fluorescein isothiocyanate. E: All cell populations were analyzed for expression of CD45RA to delineate populations of CD45RA+ and CD45RA Tconv (pseudocolor plot) and Tregs (zebra plot). F: An example of dose-response curves in Tconv and Treg subpopulations from one individual using the percentage of pSTAT5a positivity as a read out. Figure 2. Representative examples of pSTAT5a staining, the gating used to define CD4+ T-cell populations, and IL-2 dose response. Examples of pSTAT5a staining in CD4+ T cells upon stimulation with 0.1 (A), 0.25 (B) and 10 IU/mL (C) of IL-2 for 30 min, using barcoded cryopreserved PBMCs stained with CD4, CD25, CD45RA, and pSTAT5a. D: Tregs were identified using two different gating strategies: based on a high level of CD25 staining and reduced CD4 staining (CD4loCD25+ Tregs) (i) and gated on the top 2% of CD25-staining CD4+ cells (CD25hi Tregs) (ii). Tconv were identified by low/intermediate levels of CD25 staining. FITC, fluorescein isothiocyanate. E: All cell populations were analyzed for expression of CD45RA to delineate populations of CD45RA+ and CD45RA− Tconv (pseudocolor plot) and Tregs (zebra plot). F: An example of dose-response curves in Tconv and Treg subpopulations from one individual using the percentage of pSTAT5a positivity as a read out. More
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dSA triggers apoptosis and necrosis in Ins-1 cells. <em>A</em>: Qua...
Published: 13 March 2014
Figure 3 dSA triggers apoptosis and necrosis in Ins-1 cells. A: Quantification of CC-3 and p21-positive cells after 24-h incubation with 5 μmol/L SA, 1 and 5 μmol/L dSA, or BSA as control (left panel). Immunofluorescence imaging showing cytosolic expression of CC-3 (right panel). Note the pyknotic nuclei in CC-3–positive cells (arrows). Nuclei are stained with DAPI (blue). Results are mean ± SEM (n = 3). Scale bars: 50 μm. *P < 0.05. B: FACS analyses of Ins-1 cells after 24-h incubation with 1 and 5 μmol/L dSA or BSA as control and staining with PI and AnnV. C: Quantification of cells that are PI/AnnV negative (live cells), PI positive (necrotic cells), AnnV positive (apoptotic cells), and double positive. Total population comprised 20,000 cells. Note the increased lethality of cells treated with 5 μmol/L dSA. AnnV, annexin V; CC-3, cleaved caspase 3; cntl, control; dSA, 1-deoxysphinganine; FITC, fluorescein isothiocyanate; SA, sphinganine; tot, total. Figure 3. dSA triggers apoptosis and necrosis in Ins-1 cells. A: Quantification of CC-3 and p21-positive cells after 24-h incubation with 5 μmol/L SA, 1 and 5 μmol/L dSA, or BSA as control (left panel). Immunofluorescence imaging showing cytosolic expression of CC-3 (right panel). Note the pyknotic nuclei in CC-3–positive cells (arrows). Nuclei are stained with DAPI (blue). Results are mean ± SEM (n = 3). Scale bars: 50 μm. *P < 0.05. B: FACS analyses of Ins-1 cells after 24-h incubation with 1 and 5 μmol/L dSA or BSA as control and staining with PI and AnnV. C: Quantification of cells that are PI/AnnV negative (live cells), PI positive (necrotic cells), AnnV positive (apoptotic cells), and double positive. Total population comprised 20,000 cells. Note the increased lethality of cells treated with 5 μmol/L dSA. AnnV, annexin V; CC-3, cleaved caspase 3; cntl, control; dSA, 1-deoxysphinganine; FITC, fluorescein isothiocyanate; SA, sphinganine; tot, total. 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
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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
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T regulatory cells mediate long-term immune protection of NPI xenografts re...
Published: 19 January 2010
FIG. 3. T regulatory cells mediate long-term immune protection of NPI xenografts rendered by combined anti–LFA-1 and anti-CD154 mAb therapy. All normoglycemic recipients (n = 7) that received depleting anti-CD25 mAb on 0, 2, 4, and 6 days post-administration beginning at 150 days post-transplantation (arrow) became diabetic at 175.3 ± 2.5 days post-transplantation (25.3 ± 2.5 days post-injection of anti-CD25 mAb; A). NPI xenografts had infiltrating immune cells and absence of insulin-positive cells (B). Scale bar represents 100 μm. The levels of mouse anti-porcine IgG antibody (solid black line) in these mice were significantly (P < 0.0001) higher (37.28 ± 1.80%, n = 7, C) compared with the levels detected in tolerant B6 mice that were not treated with anti-CD25 mAb and in naïve nontransplanted B6 mice ( Fig. 1 N and O, respectively). Representative histograms are shown, and controls for this experiment consisted of unstained porcine spleen cells (dashed black line) and porcine spleen cells incubated with a secondary antibody without mouse serum (solid gray line). BGL, XXX; FITC, fluorescein isothiocyanate. (A high-quality color representation of this figure is available in the online issue.) FIG. 3. T regulatory cells mediate long-term immune protection of NPI xenografts rendered by combined anti–LFA-1 and anti-CD154 mAb therapy. All normoglycemic recipients (n = 7) that received depleting anti-CD25 mAb on 0, 2, 4, and 6 days post-administration beginning at 150 days post-transplantation (arrow) became diabetic at 175.3 ± 2.5 days post-transplantation (25.3 ± 2.5 days post-injection of anti-CD25 mAb; A). NPI xenografts had infiltrating immune cells and absence of insulin-positive cells (B). Scale bar represents 100 μm. The levels of mouse anti-porcine IgG antibody (solid black line) in these mice were significantly (P < 0.0001) higher (37.28 ± 1.80%, n = 7, C) compared with the levels detected in tolerant B6 mice that were not treated with anti-CD25 mAb and in naïve nontransplanted B6 mice (Fig. 1N and O, respectively). Representative histograms are shown, and controls for this experiment consisted of unstained porcine spleen cells (dashed black line) and porcine spleen cells incubated with a secondary antibody without mouse serum (solid gray line). BGL, XXX; FITC, fluorescein isothiocyanate. (A high-quality color representation of this figure is available in the online issue.) More
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Preferential recognition of posttranslationally modified peptides by T-cell...
Published: 13 April 2018
Figure 1 Preferential recognition of posttranslationally modified peptides by T-cell clones from subjects with diabetes. A: Preferential recognition was confirmed by measuring the proliferative response of T-cell clones following stimulation with either modified peptide (white squares) or the corresponding unmodified peptide (black squares), as determined by [3H] thymidine incorporation. Data are represented as stimulation index (SI) values, calculated in triplicate by normalizing the proliferation of each clone based on [3H] thymidine incorporation in unstimulated wells. Horizontal lines represent means and error bars indicate SD. Each clone exhibited negligible proliferation (SI <3) in response to unmodified peptide and robust proliferation (SI >30) in response to modified peptide. B: T-cell clones specific for each modified epitope were also stained using tetramers loaded with modified peptide (each “E” indicates a glutamic acid modification at the indicated amino acid position and each “X” indicates a citrulline modification at the indicated amino acid position) or the corresponding unmodified version (wild-type peptide [WT]), and the mean fluorescence intensities of the staining were compared. Each clone was preferentially stained by modified peptide tetramers. Results shown are representative of T-cell clones isolated from multiple subjects with type 1 diabetes (n ≥ 2). FITC, fluorescein isothiocyanate; PE, phycoerythrin; Tmer, tetramer. Figure 1. Preferential recognition of posttranslationally modified peptides by T-cell clones from subjects with diabetes. A: Preferential recognition was confirmed by measuring the proliferative response of T-cell clones following stimulation with either modified peptide (white squares) or the corresponding unmodified peptide (black squares), as determined by [3H] thymidine incorporation. Data are represented as stimulation index (SI) values, calculated in triplicate by normalizing the proliferation of each clone based on [3H] thymidine incorporation in unstimulated wells. Horizontal lines represent means and error bars indicate SD. Each clone exhibited negligible proliferation (SI <3) in response to unmodified peptide and robust proliferation (SI >30) in response to modified peptide. B: T-cell clones specific for each modified epitope were also stained using tetramers loaded with modified peptide (each “E” indicates a glutamic acid modification at the indicated amino acid position and each “X” indicates a citrulline modification at the indicated amino acid position) or the corresponding unmodified version (wild-type peptide [WT]), and the mean fluorescence intensities of the staining were compared. Each clone was preferentially stained by modified peptide tetramers. Results shown are representative of T-cell clones isolated from multiple subjects with type 1 diabetes (n ≥ 2). FITC, fluorescein isothiocyanate; PE, phycoerythrin; Tmer, tetramer. More
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Spleens and renal lymph nodes of rapamycin plus IL-10–treated mice are not ...
Published: 01 January 2006
FIG. 3. Spleens and renal lymph nodes of rapamycin plus IL-10–treated mice are not enriched in CD4+CD25+ Tr cells. A: Percentages of CD4+CD25+ T-cells in spleens and renal lymph nodes (LNs). Percentages of CD4+CD25+ (gated on CD4+ T-cells) were evaluated by FACS in spleen and renal lymph nodes of control untreated, Edmonton-treated, and rapamycin plus IL-10–treated mice. One representative experiment out of four is presented. Cy, cycrome; FITC, fluorescein isothiocyanate. B: Expression of FoxP3 in spleen and renal lymph nodes. Relative levels of mRNA FoxP3 were determined by real-time quantitative RT-PCR in total splenocytes or total renal lymph nodes of Edmonton-treated (□) and rapamycin plus IL-10–treated ( ) mice. CD4+CD25+ T-cells isolated from spleen of naïve Balb/c mice (purity ≥90%; ▒) were used as positive controls. The amounts of FoxP3 mRNA are expressed relative to that in splenocytes depleted of CD4+CD25+ T-cells (▪), which was given an arbitrary value of 1. One representative experiment out of two is presented. ***P < 0.0001 for cells vs. splenocytes depleted of CD4+CD25+ T-cells. There was no statistical significant difference between cells from Edmonton- and rapamycin plus IL-10–treated mice, The statistic refers to data in the experiment shown; statistical differences did not vary between individual experiments. FIG. 3. Spleens and renal lymph nodes of rapamycin plus IL-10–treated mice are not enriched in CD4+CD25+ Tr cells. A: Percentages of CD4+CD25+ T-cells in spleens and renal lymph nodes (LNs). Percentages of CD4+CD25+ (gated on CD4+ T-cells) were evaluated by FACS in spleen and renal lymph nodes of control untreated, Edmonton-treated, and rapamycin plus IL-10–treated mice. One representative experiment out of four is presented. Cy, cycrome; FITC, fluorescein isothiocyanate. B: Expression of FoxP3 in spleen and renal lymph nodes. Relative levels of mRNA FoxP3 were determined by real-time quantitative RT-PCR in total splenocytes or total renal lymph nodes of Edmonton-treated (□) and rapamycin plus IL-10–treated () mice. CD4+CD25+ T-cells isolated from spleen of naïve Balb/c mice (purity ≥90%; ▒) were used as positive controls. The amounts of FoxP3 mRNA are expressed relative to that in splenocytes depleted of CD4+CD25+ T-cells (▪), which was given an arbitrary value of 1. One representative experiment out of two is presented. ***P < 0.0001 for cells vs. splenocytes depleted of CD4+CD25+ T-cells. There was no statistical significant difference between cells from Edmonton- and rapamycin plus IL-10–treated mice, The statistic refers to data in the experiment shown; statistical differences did not vary between individual experiments. More