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    Size 5mL 100 reactions Price 53 0 Molecule Name dna flow cytometry reagents
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    99
    Thermo Fisher flow cytometry
    Flow Cytometry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson flow cytometry
    LTα expression during BMT is required for peripheral T‐cell reconstitution Flow <t>cytometry</t> profiles and numbers of CD4 + and CD8 + T cells (A, C) as well as CD4 + Foxp3 + Tregs (B, D) from CD45.1 donor origin in blood (A, B) and spleen (C, D) of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21, d65, and d100 upon BMT. Significance relative to WT CD45.1:WT chimeras. Histograms show numbers of CD62L + CD44 − naïve CD4 + and CD8 + T cells in the spleen of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. sjTREC were quantified by qPCR from genomic DNA of cell‐sorted splenic CD4 + and CD8 + T cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3–5 mice per group). * P
    Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 192106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore flow cytometry
    LTα expression during BMT is required for peripheral T‐cell reconstitution Flow <t>cytometry</t> profiles and numbers of CD4 + and CD8 + T cells (A, C) as well as CD4 + Foxp3 + Tregs (B, D) from CD45.1 donor origin in blood (A, B) and spleen (C, D) of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21, d65, and d100 upon BMT. Significance relative to WT CD45.1:WT chimeras. Histograms show numbers of CD62L + CD44 − naïve CD4 + and CD8 + T cells in the spleen of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. sjTREC were quantified by qPCR from genomic DNA of cell‐sorted splenic CD4 + and CD8 + T cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3–5 mice per group). * P
    Flow Cytometry, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 5842 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Becton Dickinson flow cytometric analysis
    Limited T reg cell function in the absence of developmentally established TCR specificities. (A, top) Representative flow <t>cytometric</t> analyses of lymph nodes from mice of the indicated genotypes, gated on CD4 + Foxp3 + cells. (Bottom) EGR2 staining is shown for the indicated cell populations (red, TCRβ − ; black, TCRβ + ; blue, Vα14i + ). (B–D) Lymph node cellularity (B), T cell activation status (C), and splenic T cell cytokine production (D) in Foxp3 CreERT2 Tcra WT/WT (black circles), Foxp3 CreERT2 Tcra WT/Vα14i-StopF (blue circles), Foxp3 CreERT2 Tcra FL/FL (red circles), and Foxp3 CreERT2 Tcra FL/Vα14i-StopF (gray circles) mice. Each circle represents an individual mouse. P-values were calculated using unpaired Student’s t test. **, P
    Flow Cytometric Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 96/100, based on 42751 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FlowJo flow cytometry data
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry Data, supplied by FlowJo, used in various techniques. Bioz Stars score: 93/100, based on 11467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec flow cytometry
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 2675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher flow cytometry analysis
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend flow cytometry
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 2755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Beckman Coulter flow cytometry
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 99/100, based on 31064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies flow cytometry
    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow <t>cytometry.</t> This experiment was performed twice.
    Flow Cytometry, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 1871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore flow cytometry analysis
    hTERT expression in CSCs is mutually exclusive with the mesenchymal phenotype. (A) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) showing that the loss of mesenchymal phenotype in hTERT high CSCs mediated by PD173074 is associated with the loss of hTERT expression. However, acquisition of a mesenchymal phenotype in hTERT -/low CSCs mediated by TGF-β is associated with increased hTERT expression. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Flow <t>cytometry</t> overlay histogram analysis of N-cadherin, E-cadherin and hTERT showing that hTERT high CSCs treated with PD173074 lose their mesenchymal phenotype, which was associated with a loss of hTERT expression. Additionally, hTERT -/low CSCs treated with TGF-β acquired a mesenchymal phenotype, which was associated with increased hTERT expression. For comparison, an isotype control was used to define the positive and negative population for each marker.
    Flow Cytometry Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer set
    hTERT expression in CSCs is mutually exclusive with the mesenchymal phenotype. (A) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) showing that the loss of mesenchymal phenotype in hTERT high CSCs mediated by PD173074 is associated with the loss of hTERT expression. However, acquisition of a mesenchymal phenotype in hTERT -/low CSCs mediated by TGF-β is associated with increased hTERT expression. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Flow <t>cytometry</t> overlay histogram analysis of N-cadherin, E-cadherin and hTERT showing that hTERT high CSCs treated with PD173074 lose their mesenchymal phenotype, which was associated with a loss of hTERT expression. Additionally, hTERT -/low CSCs treated with TGF-β acquired a mesenchymal phenotype, which was associated with increased hTERT expression. For comparison, an isotype control was used to define the positive and negative population for each marker.
    Buffer Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FlowJo flow cytometric data
    The autoimmune phenotype of Roquin san/san mice requires T cell activation through CD28, and T FH cells are expanded cell autonomously. (a, left) Detection of IgG-ANA using Hep-2 slides in sera from 8-wk-old female Roquin san/san and Roquin san/san Cd28 −/− mice ( n = 5 per group). (right) Detection of anti-dsDNA IgG serum antibodies in 6-mo-old female Roquin san/san and Roquin san/san Cd28 −/− mice determined by staining C. luciliae slides. Data are representative of three independent experiments ( n ≥ 5 per group). (b) Score of nephritis severity in 6-mo-old female Roquin +/+ Roquin san/san and Roquin san/san Cd28 −/− mice as determined by histological analysis defined by the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Each symbol represents one mouse. Horizontal bars indicate medians. (c) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right). Histology from Roquin san/san Cd28 −/− animals was much less severe, with normal H E appearances and few electron-dense deposits (arrows) in the mesangium. Bars: (left) 100 µm; (right) 10 µm. (d) Representative flow <t>cytometric</t> contour plots of CD4 + CXCR5 + PD-1 high T FH cells in 10-wk-old Roquin san/san mice and control littermates. Data are representative of five independent experiments ( n = 4 per group), and the numbers in the plots represent percentages. (e) Dot plots representing percentages of CD4 + CXCR5 + PD-1 high T FH cells from SRBC-immunized chimeric mice generated by reconstituting sublethally irradiated mice with a 1:1 mix of either Roquin +/+ .Ly5 a / Roquin +/+ .Ly5 b (left) or Roquin +/+ .Ly5 a / Roquin san/san .Ly5 b (right). Data are representative of three independent experiments ( n = 4 per group). Each symbol represents the Ly5 a or Ly5 b population derived from one mouse. (f) ELISA was used to determine culture supernatant IL-21 levels from 24-h splenocyte cultures from Roquin san/san mice and littermate controls in the presence of PMA and ionomycin. Error bars indicate means ± SEM. Data are representative of three independent tests where each sample was run in triplicate.
    Flow Cytometric Data, supplied by FlowJo, used in various techniques. Bioz Stars score: 92/100, based on 2805 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam flow cytometry
    The autoimmune phenotype of Roquin san/san mice requires T cell activation through CD28, and T FH cells are expanded cell autonomously. (a, left) Detection of IgG-ANA using Hep-2 slides in sera from 8-wk-old female Roquin san/san and Roquin san/san Cd28 −/− mice ( n = 5 per group). (right) Detection of anti-dsDNA IgG serum antibodies in 6-mo-old female Roquin san/san and Roquin san/san Cd28 −/− mice determined by staining C. luciliae slides. Data are representative of three independent experiments ( n ≥ 5 per group). (b) Score of nephritis severity in 6-mo-old female Roquin +/+ Roquin san/san and Roquin san/san Cd28 −/− mice as determined by histological analysis defined by the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Each symbol represents one mouse. Horizontal bars indicate medians. (c) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right). Histology from Roquin san/san Cd28 −/− animals was much less severe, with normal H E appearances and few electron-dense deposits (arrows) in the mesangium. Bars: (left) 100 µm; (right) 10 µm. (d) Representative flow <t>cytometric</t> contour plots of CD4 + CXCR5 + PD-1 high T FH cells in 10-wk-old Roquin san/san mice and control littermates. Data are representative of five independent experiments ( n = 4 per group), and the numbers in the plots represent percentages. (e) Dot plots representing percentages of CD4 + CXCR5 + PD-1 high T FH cells from SRBC-immunized chimeric mice generated by reconstituting sublethally irradiated mice with a 1:1 mix of either Roquin +/+ .Ly5 a / Roquin +/+ .Ly5 b (left) or Roquin +/+ .Ly5 a / Roquin san/san .Ly5 b (right). Data are representative of three independent experiments ( n = 4 per group). Each symbol represents the Ly5 a or Ly5 b population derived from one mouse. (f) ELISA was used to determine culture supernatant IL-21 levels from 24-h splenocyte cultures from Roquin san/san mice and littermate controls in the presence of PMA and ionomycin. Error bars indicate means ± SEM. Data are representative of three independent tests where each sample was run in triplicate.
    Flow Cytometry, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen flow cytometry
    The autoimmune phenotype of Roquin san/san mice requires T cell activation through CD28, and T FH cells are expanded cell autonomously. (a, left) Detection of IgG-ANA using Hep-2 slides in sera from 8-wk-old female Roquin san/san and Roquin san/san Cd28 −/− mice ( n = 5 per group). (right) Detection of anti-dsDNA IgG serum antibodies in 6-mo-old female Roquin san/san and Roquin san/san Cd28 −/− mice determined by staining C. luciliae slides. Data are representative of three independent experiments ( n ≥ 5 per group). (b) Score of nephritis severity in 6-mo-old female Roquin +/+ Roquin san/san and Roquin san/san Cd28 −/− mice as determined by histological analysis defined by the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Each symbol represents one mouse. Horizontal bars indicate medians. (c) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right). Histology from Roquin san/san Cd28 −/− animals was much less severe, with normal H E appearances and few electron-dense deposits (arrows) in the mesangium. Bars: (left) 100 µm; (right) 10 µm. (d) Representative flow <t>cytometric</t> contour plots of CD4 + CXCR5 + PD-1 high T FH cells in 10-wk-old Roquin san/san mice and control littermates. Data are representative of five independent experiments ( n = 4 per group), and the numbers in the plots represent percentages. (e) Dot plots representing percentages of CD4 + CXCR5 + PD-1 high T FH cells from SRBC-immunized chimeric mice generated by reconstituting sublethally irradiated mice with a 1:1 mix of either Roquin +/+ .Ly5 a / Roquin +/+ .Ly5 b (left) or Roquin +/+ .Ly5 a / Roquin san/san .Ly5 b (right). Data are representative of three independent experiments ( n = 4 per group). Each symbol represents the Ly5 a or Ly5 b population derived from one mouse. (f) ELISA was used to determine culture supernatant IL-21 levels from 24-h splenocyte cultures from Roquin san/san mice and littermate controls in the presence of PMA and ionomycin. Error bars indicate means ± SEM. Data are representative of three independent tests where each sample was run in triplicate.
    Flow Cytometry, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson facscalibur flow cytometry
    OA-induced activation of PDHs through the regulation of · O 2 − levels by SIRT3. ( a ) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The levels of cellular superoxide anion ( · O 2 − ) and hydrogen peroxide (H 2 O 2 ) were detected by <t>FACSCalibur</t> flow <t>cytometry</t> using the fluorescent dye dihydroethidium at Ex/Em of 300/610 nm or dichlorofluorescein-diacetate at Ex/Em of 488/525 nm, respectively. ( b ) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μ M OA for 10 h under hypoxia, and · O 2 − was detected. ( c , d ) Cells were treated with 100 μ M OA or 10 mM N -acetyl cysteine (antioxidant used as a positive control) in the presence of 1 μ M rotenone for 10 h under hypoxia. The levels of · O 2 − ( c ), protein expression of HIF-OH and HIF1 α ( d ) were detected. ( e , f ) Cells were treated with 100 μ M OA with/without 1 μ M rotenone for 10 h under hypoxia. Cell survival rate ( e ), glucose uptake and production of lactic acid ( f ) were detected, respectively. Bars, S.D.; * P
    Facscalibur Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson facscan flow cytometry
    Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by propidium iodide were detected using <t>FACScan</t> flow <t>cytometry.</t> The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P
    Facscan Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2628 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore apoptosis
    Protective effects of eckol, dieckol and 8,8′-bieckol on Aβ 25-35 -indued <t>apoptosis</t> in PC12 cells. ( A ) Morphological apoptosis determined by fluorescence microscopy (400×). ( B ) The percentage of apoptotic cells of the total number of cells. ( C , D ) Early and late apoptosis detected by flow cytometry using Annexin V/7-AAD staining. The lower left area means normal cells (annexin V−/7-AAD−), lower right area means early apoptotic cells (annexin V+/7-AAD−), upper right area means late apopotic and dead cells (annexin V+/7-AAD+) and upper left area means dead cells (annexin V−/7-AAD+). ( E ) Analysis of MMP examined by rhodamine 123. ( F ) The intracellular Ca 2+ levels analyzed using fluo-3AM. ### p
    Apoptosis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Partec flow cytometry
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    Protective effects of eckol, dieckol and 8,8′-bieckol on Aβ 25-35 -indued <t>apoptosis</t> in PC12 cells. ( A ) Morphological apoptosis determined by fluorescence microscopy (400×). ( B ) The percentage of apoptotic cells of the total number of cells. ( C , D ) Early and late apoptosis detected by flow cytometry using Annexin V/7-AAD staining. The lower left area means normal cells (annexin V−/7-AAD−), lower right area means early apoptotic cells (annexin V+/7-AAD−), upper right area means late apopotic and dead cells (annexin V+/7-AAD+) and upper left area means dead cells (annexin V−/7-AAD+). ( E ) Analysis of MMP examined by rhodamine 123. ( F ) The intracellular Ca 2+ levels analyzed using fluo-3AM. ### p
    Flow Cytometry, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 807 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LTα expression during BMT is required for peripheral T‐cell reconstitution Flow cytometry profiles and numbers of CD4 + and CD8 + T cells (A, C) as well as CD4 + Foxp3 + Tregs (B, D) from CD45.1 donor origin in blood (A, B) and spleen (C, D) of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21, d65, and d100 upon BMT. Significance relative to WT CD45.1:WT chimeras. Histograms show numbers of CD62L + CD44 − naïve CD4 + and CD8 + T cells in the spleen of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. sjTREC were quantified by qPCR from genomic DNA of cell‐sorted splenic CD4 + and CD8 + T cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3–5 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: LTα expression during BMT is required for peripheral T‐cell reconstitution Flow cytometry profiles and numbers of CD4 + and CD8 + T cells (A, C) as well as CD4 + Foxp3 + Tregs (B, D) from CD45.1 donor origin in blood (A, B) and spleen (C, D) of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21, d65, and d100 upon BMT. Significance relative to WT CD45.1:WT chimeras. Histograms show numbers of CD62L + CD44 − naïve CD4 + and CD8 + T cells in the spleen of WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. sjTREC were quantified by qPCR from genomic DNA of cell‐sorted splenic CD4 + and CD8 + T cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d21 pBMT. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3–5 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Real-time Polymerase Chain Reaction

    In vitro stimulation with RANKL induces the upregulation of LTα specifically in thymic LTi cells LTα protein expression was analyzed by flow cytometry in thymic LTi cells from sublethally irradiated WT mice treated in vitro for 24 h with GST, RANKL‐GST, or RANKL‐GST + RANK‐Fc. The histogram shows the MFI of LTα for each condition. Iso: Isotype control. LTα protein was analyzed in DN, DP, CD4 + , and CD8 + SP as well as in LTi cells purified from sublethally irradiated WT mice and treated in vitro for 24 h with GST or RANKL‐GST. Results are represented as fold change relative to the GST condition. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: In vitro stimulation with RANKL induces the upregulation of LTα specifically in thymic LTi cells LTα protein expression was analyzed by flow cytometry in thymic LTi cells from sublethally irradiated WT mice treated in vitro for 24 h with GST, RANKL‐GST, or RANKL‐GST + RANK‐Fc. The histogram shows the MFI of LTα for each condition. Iso: Isotype control. LTα protein was analyzed in DN, DP, CD4 + , and CD8 + SP as well as in LTi cells purified from sublethally irradiated WT mice and treated in vitro for 24 h with GST or RANKL‐GST. Results are represented as fold change relative to the GST condition. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: In Vitro, Expressing, Flow Cytometry, Cytometry, Irradiation, Mouse Assay, Purification

    RANKL is upregulated in CD 4 + SP and LT i cells during the course of thymic regeneration Expression of RANKL protein analyzed by flow cytometry in CD45 − and CD45 + thymic cells from untreated (UT) WT mice or at d3 SL‐TBI. Flow cytometry profiles and frequencies of DN (double negative), DP (double positive), CD4 + and CD8 + SP (single positive) (B), and LTi cells (C) from untreated (UT) WT mice or at d3 SL‐TBI. Expression level of RANKL protein in CD4 + SP and LTi cells from UT WT mice or at d3 SL‐TBI and L‐TBI. Expression of Rankl mRNA in the total thymus isolated from UT WT, Rorc −/− , ZAP‐70 −/− , and Rag2 −/− mice or at d3 SL‐TBI ( n = 3–6 mice per genotype). CD4 + SP and LTi cells from UT WT mice or at d3, d6, d10, and d20 SL‐TBI with no hematopoietic rescue were analyzed for the expression of RANKL protein. Mean fluorescence intensity (MFI) of RANKL in CD4 + SP and LTi cells over time following SL‐TBI. The red lines represent the MFI of RANKL at baseline. Data information: Data are shown as mean ± SEM and are pooled of four independent experiments with similar results ( n = 3–4 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: RANKL is upregulated in CD 4 + SP and LT i cells during the course of thymic regeneration Expression of RANKL protein analyzed by flow cytometry in CD45 − and CD45 + thymic cells from untreated (UT) WT mice or at d3 SL‐TBI. Flow cytometry profiles and frequencies of DN (double negative), DP (double positive), CD4 + and CD8 + SP (single positive) (B), and LTi cells (C) from untreated (UT) WT mice or at d3 SL‐TBI. Expression level of RANKL protein in CD4 + SP and LTi cells from UT WT mice or at d3 SL‐TBI and L‐TBI. Expression of Rankl mRNA in the total thymus isolated from UT WT, Rorc −/− , ZAP‐70 −/− , and Rag2 −/− mice or at d3 SL‐TBI ( n = 3–6 mice per genotype). CD4 + SP and LTi cells from UT WT mice or at d3, d6, d10, and d20 SL‐TBI with no hematopoietic rescue were analyzed for the expression of RANKL protein. Mean fluorescence intensity (MFI) of RANKL in CD4 + SP and LTi cells over time following SL‐TBI. The red lines represent the MFI of RANKL at baseline. Data information: Data are shown as mean ± SEM and are pooled of four independent experiments with similar results ( n = 3–4 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Isolation, Fluorescence

    LT α is critical for TEC regeneration during the course of BMT Expression level of LTβR protein in total TECs, cTECs, mTECs, and TEPC‐enriched cells from the thymus of UT WT mice ( n = 6) and at d3 SL‐TBI ( n = 6) was analyzed by flow cytometry. FMO: Fluorescence Minus One. Flow cytometry profiles and numbers of total TECs (B); cTECs, mTECs (C); mTEC subsets (D); cTEC hi , mTEC hi , TEC lo , mTEC lo (E); and TEPC‐enriched cells (F) were analyzed in CD45 neg ‐enriched cells by AutoMACS from the thymus of UT WT and LTα −/− mice or in WT CD45.1:WT and WT CD45.1:LTα −/− chimeras at d10, d21, and d65 upon BMT. Numbers of total proliferating Ki‐67 + TECs, cTECs, mTECs, and TEPC‐enriched cells at the indicated time points. The expression of mRNAs coding for Aire , Aire ‐induced TRAs ( Sp1 and Sp2 ); Aire ‐independent TRA ( casein β ); Fezf2 and Fezf2 ‐induced TRAs ( Apoc3 , Fabp9, and Resp18 ) was measured by qPCR in CD45 − thymic stromal cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d65 after BMT. Significance relative to WT CD45.1:WT chimeras. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: LT α is critical for TEC regeneration during the course of BMT Expression level of LTβR protein in total TECs, cTECs, mTECs, and TEPC‐enriched cells from the thymus of UT WT mice ( n = 6) and at d3 SL‐TBI ( n = 6) was analyzed by flow cytometry. FMO: Fluorescence Minus One. Flow cytometry profiles and numbers of total TECs (B); cTECs, mTECs (C); mTEC subsets (D); cTEC hi , mTEC hi , TEC lo , mTEC lo (E); and TEPC‐enriched cells (F) were analyzed in CD45 neg ‐enriched cells by AutoMACS from the thymus of UT WT and LTα −/− mice or in WT CD45.1:WT and WT CD45.1:LTα −/− chimeras at d10, d21, and d65 upon BMT. Numbers of total proliferating Ki‐67 + TECs, cTECs, mTECs, and TEPC‐enriched cells at the indicated time points. The expression of mRNAs coding for Aire , Aire ‐induced TRAs ( Sp1 and Sp2 ); Aire ‐independent TRA ( casein β ); Fezf2 and Fezf2 ‐induced TRAs ( Apoc3 , Fabp9, and Resp18 ) was measured by qPCR in CD45 − thymic stromal cells from WT CD45.1:WT and WT CD45.1:LTα −/− mice at d65 after BMT. Significance relative to WT CD45.1:WT chimeras. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Real-time Polymerase Chain Reaction

    RANKL is crucially involved in TEC regeneration after TBI Flow cytometry profiles and numbers of total TECs (EpCAM + ), cTECs (UEA‐1 − Ly51 + ), mTECs (UEA‐1 + Ly51 − ), and mTEC subsets (CD80 lo Aire − , CD80 hi Aire − , and CD80 hi Aire + ) analyzed in CD45 neg ‐enriched cells by AutoMACS from UT WT mice or treated with PBS, a RANKL isotype control antibody (Iso.), a neutralizing anti‐RANKL antibody (IK22/5), GST or RANKL proteins during 3 days upon SL‐TBI. Histograms show numbers of cTEC hi (MHCII hi UEA‐1 − ), mTEC hi (MHCII hi UEA‐1 + ), TEC lo (MHCII lo UEA‐1 − ), mTEC lo (MHCII lo UEA‐1 + ) (B); TEPC‐enriched cells (defined as α6‐integrin hi Sca‐1 hi in the TEC lo subset) (C); and proliferating Ki‐67 + cTECs, mTECs, and TEPC‐enriched cells (D). Expression of mRNAs coding for pro‐ ( Bax, Bid, Bak ) and anti‐apoptotic ( Bcl‐xl ) proteins analyzed by qPCR in purified cTECs and mTECs. Thymic sections from WT mice treated with GST or RANKL during 3 days upon SL‐TBI were stained for the expression of K14 and Aire. The histogram shows the density of Aire + cells in medullary area. m denotes the medulla. Fifteen sections were quantified for each condition; scale bar: 100 μm. The expression of Aire and TRAs ( Sp1 and Sp2 ) in purified mTECs (G) and Selp, Icam‐1, Ccl19, and Ccl21 in purified cTECs (H) from WT mice treated with GST or RANKL was analyzed by qPCR. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–4 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: RANKL is crucially involved in TEC regeneration after TBI Flow cytometry profiles and numbers of total TECs (EpCAM + ), cTECs (UEA‐1 − Ly51 + ), mTECs (UEA‐1 + Ly51 − ), and mTEC subsets (CD80 lo Aire − , CD80 hi Aire − , and CD80 hi Aire + ) analyzed in CD45 neg ‐enriched cells by AutoMACS from UT WT mice or treated with PBS, a RANKL isotype control antibody (Iso.), a neutralizing anti‐RANKL antibody (IK22/5), GST or RANKL proteins during 3 days upon SL‐TBI. Histograms show numbers of cTEC hi (MHCII hi UEA‐1 − ), mTEC hi (MHCII hi UEA‐1 + ), TEC lo (MHCII lo UEA‐1 − ), mTEC lo (MHCII lo UEA‐1 + ) (B); TEPC‐enriched cells (defined as α6‐integrin hi Sca‐1 hi in the TEC lo subset) (C); and proliferating Ki‐67 + cTECs, mTECs, and TEPC‐enriched cells (D). Expression of mRNAs coding for pro‐ ( Bax, Bid, Bak ) and anti‐apoptotic ( Bcl‐xl ) proteins analyzed by qPCR in purified cTECs and mTECs. Thymic sections from WT mice treated with GST or RANKL during 3 days upon SL‐TBI were stained for the expression of K14 and Aire. The histogram shows the density of Aire + cells in medullary area. m denotes the medulla. Fifteen sections were quantified for each condition; scale bar: 100 μm. The expression of Aire and TRAs ( Sp1 and Sp2 ) in purified mTECs (G) and Selp, Icam‐1, Ccl19, and Ccl21 in purified cTECs (H) from WT mice treated with GST or RANKL was analyzed by qPCR. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–4 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Purification, Staining

    The critical role of LTα in thymic regeneration and peripheral T‐cell reconstitution persists with age CD4 + CD8 − thymic cells from 8‐month‐old UT WT mice ( n = 9) or at d3 SL‐TBI ( n = 6) were analyzed for the expression of RORγt by flow cytometry. MFI of LTβR‐Fc staining in thymic LTi cells from 6‐ to 8‐month‐old UT WT mice or at d3 SL‐TBI. Histograms show numbers of total TECs, cTECs, mTECs (C); Ki‐67 + TEC subsets (D); mTEC subsets (E); total thymic cells, T‐cell subsets (DN, DP, CD4 + SP, and CD8 + SP) (F); and ETPs (G) in the thymus from WT CD45.1:WT and WT CD45.1:LTα −/− chimeras of 6–8 months of age at d21 upon BMT. Flow cytometry profiles and numbers of CD4 + and CD8 + T cells (H, J) as well as CD4 + Foxp3 + Tregs (I, K) from CD45.1 donor origin in blood (H, I) and spleen (J, K) of WT CD45.1:WT and WT CD45.1:LTα −/− mice of 6–8 months of age at d21 pBMT. Significance relative to WT CD45.1:WT chimeras. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: The critical role of LTα in thymic regeneration and peripheral T‐cell reconstitution persists with age CD4 + CD8 − thymic cells from 8‐month‐old UT WT mice ( n = 9) or at d3 SL‐TBI ( n = 6) were analyzed for the expression of RORγt by flow cytometry. MFI of LTβR‐Fc staining in thymic LTi cells from 6‐ to 8‐month‐old UT WT mice or at d3 SL‐TBI. Histograms show numbers of total TECs, cTECs, mTECs (C); Ki‐67 + TEC subsets (D); mTEC subsets (E); total thymic cells, T‐cell subsets (DN, DP, CD4 + SP, and CD8 + SP) (F); and ETPs (G) in the thymus from WT CD45.1:WT and WT CD45.1:LTα −/− chimeras of 6–8 months of age at d21 upon BMT. Flow cytometry profiles and numbers of CD4 + and CD8 + T cells (H, J) as well as CD4 + Foxp3 + Tregs (I, K) from CD45.1 donor origin in blood (H, I) and spleen (J, K) of WT CD45.1:WT and WT CD45.1:LTα −/− mice of 6–8 months of age at d21 pBMT. Significance relative to WT CD45.1:WT chimeras. Data information: Data are shown as mean ± SEM and are pooled of two independent experiments with similar results ( n = 3 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Staining

    TECs but not fibroblasts and LTi cells are severely reduced in LTα −/− mice at d3 SL‐TBI Histograms show numbers of cTECs and mTECs (A) as well as mTEC subsets (B) in WT and LTα −/− mice at d3 SL‐TBI. Thymic sections from WT and LTα −/− mice at d3 SL‐TBI were stained for the expression of K14 and Aire. The histogram shows the density of Aire + cells in medullary area. m, medulla. Fifteen sections were quantified; scale bar: 100 μm. The histogram shows numbers of CD45 − PDFRα + fibroblasts in WT and LTα −/− mice at d3 SL‐TBI. Flow cytometry profiles and frequencies of thymic LTi cells from WT or LTα −/− mice at d3 SL‐TBI. Data information: Data are shown as mean ± SEM and are pooled of five independent experiments with similar results ( n = 3 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: TECs but not fibroblasts and LTi cells are severely reduced in LTα −/− mice at d3 SL‐TBI Histograms show numbers of cTECs and mTECs (A) as well as mTEC subsets (B) in WT and LTα −/− mice at d3 SL‐TBI. Thymic sections from WT and LTα −/− mice at d3 SL‐TBI were stained for the expression of K14 and Aire. The histogram shows the density of Aire + cells in medullary area. m, medulla. Fifteen sections were quantified; scale bar: 100 μm. The histogram shows numbers of CD45 − PDFRα + fibroblasts in WT and LTα −/− mice at d3 SL‐TBI. Flow cytometry profiles and frequencies of thymic LTi cells from WT or LTα −/− mice at d3 SL‐TBI. Data information: Data are shown as mean ± SEM and are pooled of five independent experiments with similar results ( n = 3 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Mouse Assay, Staining, Expressing, Flow Cytometry, Cytometry

    LT α is required for de novo thymopoiesis during BMT Numbers of total thymic cells and thymocyte subsets of CD45.1 donor origin were analyzed by flow cytometry in WT CD45.1:WT and WT CD45.1:LTα −/− mice at d10, d21, and d65 after BMT. Numbers of DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ), and DN4 (CD44 − CD25 − ) of CD45.1 origin were also analyzed at d10 and d21 after BMT. Numbers of early thymic progenitors (ETPs; CD4 − CD8 − CD44 + CD25 − Lin − CD117 + ) and BM prethymic progenitors (CD3 − CD44 + CD25 − Lin − CD117 + ) were analyzed by flow cytometry in UT WT and LTα −/− mice or WT CD45.1:WT and WT CD45.1:LTα −/− chimeras at d65 after BMT. The expression of Ccl19, Ccl21, Ccl25, Icam‐1 , Vcam‐1, and Selp mRNAs was measured by qPCR in purified EpCAM + TECs, CD31 + EpCAM − endothelial cells, and gp38 + EpCAM − fibroblasts isolated from WT CD45.1:WT ( n = 5) and WT CD45.1:LTα −/− ( n = 5) chimeras at d21 after BMT. Numbers of ETPs and total cells of CD45.1 donor origin in CD45.2 WT or LTα −/− recipients 48 h after i.v. injection of CD45.1 BM cells. Expression of Il‐22 mRNA in the total thymus isolated from UT WT and LTα −/− mice ( n = 4) or at d3 SL‐TBI ( n = 4). Expression of Il‐23 mRNA in the total thymus and purified DCs isolated from UT WT and LTα −/− mice ( n = 4) or at d3 SL‐TBI ( n = 4). Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: LT α is required for de novo thymopoiesis during BMT Numbers of total thymic cells and thymocyte subsets of CD45.1 donor origin were analyzed by flow cytometry in WT CD45.1:WT and WT CD45.1:LTα −/− mice at d10, d21, and d65 after BMT. Numbers of DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ), and DN4 (CD44 − CD25 − ) of CD45.1 origin were also analyzed at d10 and d21 after BMT. Numbers of early thymic progenitors (ETPs; CD4 − CD8 − CD44 + CD25 − Lin − CD117 + ) and BM prethymic progenitors (CD3 − CD44 + CD25 − Lin − CD117 + ) were analyzed by flow cytometry in UT WT and LTα −/− mice or WT CD45.1:WT and WT CD45.1:LTα −/− chimeras at d65 after BMT. The expression of Ccl19, Ccl21, Ccl25, Icam‐1 , Vcam‐1, and Selp mRNAs was measured by qPCR in purified EpCAM + TECs, CD31 + EpCAM − endothelial cells, and gp38 + EpCAM − fibroblasts isolated from WT CD45.1:WT ( n = 5) and WT CD45.1:LTα −/− ( n = 5) chimeras at d21 after BMT. Numbers of ETPs and total cells of CD45.1 donor origin in CD45.2 WT or LTα −/− recipients 48 h after i.v. injection of CD45.1 BM cells. Expression of Il‐22 mRNA in the total thymus isolated from UT WT and LTα −/− mice ( n = 4) or at d3 SL‐TBI ( n = 4). Expression of Il‐23 mRNA in the total thymus and purified DCs isolated from UT WT and LTα −/− mice ( n = 4) or at d3 SL‐TBI ( n = 4). Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Purification, Isolation, Injection

    RANKL boosts TEC regeneration and de novo thymopoiesis in an LT α‐dependent manner upon BMT Experimental setup: WT CD45.1:WT and WT CD45.1:LTα −/− chimeras were treated with GST or RANKL‐GST proteins at d2, d4, and d6 after BMT and TEC regeneration and T‐cell reconstitution were analyzed at d21 after BMT. Expression level of LTα protein in thymic LTi cells in UT mice or treated with GST or RANKL‐GST. Thymic sections from WT CD45.1:WT and WT CD45.1:LTα −/− mice treated with GST and RANKL at d2, d4, and d6 after BMT were stained for the expression of K14 at d21 pBMT. The histogram shows quantifications of medullary areas. m and c denote the medulla and the cortex, respectively. Twenty sections were quantified for each condition; scale bar: 100 μm. Numbers of total TECs, cTECs, and mTECs (D) and flow cytometry profiles of Aire + mTECs in total EpCAM + TECs (E). Expression of mRNAs coding for TRAs ( Sp1 and Sp2 ) in CD45?thymic stromal cells analyzed by qPCR. Numbers of total cells and thymocyte subsets of CD45.1 donor origin analyzed in the thymus. Numbers of ETPs of CD45.1 donor origin in the thymus and prethymic progenitors in the BM from CD45.2 WT or LTα −/− recipients 48 h after i.v. injection of CD45.1 BM cells. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: RANKL boosts TEC regeneration and de novo thymopoiesis in an LT α‐dependent manner upon BMT Experimental setup: WT CD45.1:WT and WT CD45.1:LTα −/− chimeras were treated with GST or RANKL‐GST proteins at d2, d4, and d6 after BMT and TEC regeneration and T‐cell reconstitution were analyzed at d21 after BMT. Expression level of LTα protein in thymic LTi cells in UT mice or treated with GST or RANKL‐GST. Thymic sections from WT CD45.1:WT and WT CD45.1:LTα −/− mice treated with GST and RANKL at d2, d4, and d6 after BMT were stained for the expression of K14 at d21 pBMT. The histogram shows quantifications of medullary areas. m and c denote the medulla and the cortex, respectively. Twenty sections were quantified for each condition; scale bar: 100 μm. Numbers of total TECs, cTECs, and mTECs (D) and flow cytometry profiles of Aire + mTECs in total EpCAM + TECs (E). Expression of mRNAs coding for TRAs ( Sp1 and Sp2 ) in CD45?thymic stromal cells analyzed by qPCR. Numbers of total cells and thymocyte subsets of CD45.1 donor origin analyzed in the thymus. Numbers of ETPs of CD45.1 donor origin in the thymus and prethymic progenitors in the BM from CD45.2 WT or LTα −/− recipients 48 h after i.v. injection of CD45.1 BM cells. Data information: Data are shown as mean ± SEM and are pooled of three independent experiments with similar results ( n = 3–5 mice per group). * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Injection

    RANKL administration induces LT α upregulation specifically in thymic LT i cells after TBI Expression of RANK receptor in thymic LTi cells from UT WT ( n = 6) mice and at d3 SL‐TBI ( n = 6). Expression level of LTα protein in thymic LTi cells from WT mice treated in vivo with PBS ( n = 6), GST ( n = 6), or RANKL‐GST ( n = 6) during 3 days after SL‐TBI. LTα protein was analyzed in thymocyte subsets and LTi cells from WT mice treated in vivo with GST ( n = 9) or RANKL‐GST ( n = 9) during 3 days after SL‐TBI. Results are represented as fold change relative to the GST condition. Data are pooled of three experiments. Expression level of LTα protein in thymic LTi cells from WT mice treated in vivo with PBS ( n = 6), an isotype control (Iso.) ( n = 3), or a neutralizing anti‐RANKL antibody (IL22/5) ( n = 6) during 3 days after SL‐TBI. LTα protein was analyzed in thymocyte subsets and LTi cells from WT mice treated in vivo with an isotype control ( n = 3), or a neutralizing anti‐RANKL antibody ( n = 6) during 3 days after SL‐TBI. Results are represented as fold change relative to the isotype condition. Expression of Ltα mRNA in the total thymus isolated from UT WT, Rorc −/− , and ZAP‐70 −/− mice or at d3 SL‐TBI ( n = 3–6 mice per genotype). Expression of Ltα mRNA in the total thymus isolated from irradiated: WT, Rag2 −/− mice, and WT mice treated 3 days before with dexamethasone (Dexa). Data are pooled of two to three experiments ( n = 6–12 mice per group). Correlation of RANKL and LTα expression in thymic LTi cells during the course of BMT. pBMT: post‐bone marrow transplantation. Data are pooled of three independent experiments with similar results ( n = 3–4 mice per group). Expression level of LTα protein in thymic LTi cells from UT WT mice or at d3, d6, d10, and d21 after BMT. Data are pooled of three independent experiments with similar results ( n = 3–4 mice per group). Expression level of LTα protein analyzed by flow cytometry in thymic LTi cells from CD45.1 donor and CD45.2 host origin at d3 and d6 after BMT. Data are pooled of four experiments ( n = 3–4 mice per group). RANKL protein expression in LTi cells from WT and LTα −/− mice at d3 SL‐TBI. Data are pooled of three experiments ( n = 3–5 mice per group). The expression of Ltα and Ltβ mRNAs was measured by qPCR in the total thymus from UT WT mice ( n = 4) or at d3 SL‐TBI ( n = 4). MFI of LTα protein in CD45 − and CD45 + thymic cells from UT WT ( n = 6) mice or at d3 SL‐TBI ( n = 6). Representative histogram of LTα (N) and LTβR‐Fc staining (O) in LTi cells from UT WT mice ( n = 6) or at d3 SL‐TBI ( n = 6) or L‐TBI ( n = 6). Data information: Data are shown as mean ± SEM. * P

    Journal: EMBO Molecular Medicine

    Article Title: Administration of RANKL boosts thymic regeneration upon bone marrow transplantation

    doi: 10.15252/emmm.201607176

    Figure Lengend Snippet: RANKL administration induces LT α upregulation specifically in thymic LT i cells after TBI Expression of RANK receptor in thymic LTi cells from UT WT ( n = 6) mice and at d3 SL‐TBI ( n = 6). Expression level of LTα protein in thymic LTi cells from WT mice treated in vivo with PBS ( n = 6), GST ( n = 6), or RANKL‐GST ( n = 6) during 3 days after SL‐TBI. LTα protein was analyzed in thymocyte subsets and LTi cells from WT mice treated in vivo with GST ( n = 9) or RANKL‐GST ( n = 9) during 3 days after SL‐TBI. Results are represented as fold change relative to the GST condition. Data are pooled of three experiments. Expression level of LTα protein in thymic LTi cells from WT mice treated in vivo with PBS ( n = 6), an isotype control (Iso.) ( n = 3), or a neutralizing anti‐RANKL antibody (IL22/5) ( n = 6) during 3 days after SL‐TBI. LTα protein was analyzed in thymocyte subsets and LTi cells from WT mice treated in vivo with an isotype control ( n = 3), or a neutralizing anti‐RANKL antibody ( n = 6) during 3 days after SL‐TBI. Results are represented as fold change relative to the isotype condition. Expression of Ltα mRNA in the total thymus isolated from UT WT, Rorc −/− , and ZAP‐70 −/− mice or at d3 SL‐TBI ( n = 3–6 mice per genotype). Expression of Ltα mRNA in the total thymus isolated from irradiated: WT, Rag2 −/− mice, and WT mice treated 3 days before with dexamethasone (Dexa). Data are pooled of two to three experiments ( n = 6–12 mice per group). Correlation of RANKL and LTα expression in thymic LTi cells during the course of BMT. pBMT: post‐bone marrow transplantation. Data are pooled of three independent experiments with similar results ( n = 3–4 mice per group). Expression level of LTα protein in thymic LTi cells from UT WT mice or at d3, d6, d10, and d21 after BMT. Data are pooled of three independent experiments with similar results ( n = 3–4 mice per group). Expression level of LTα protein analyzed by flow cytometry in thymic LTi cells from CD45.1 donor and CD45.2 host origin at d3 and d6 after BMT. Data are pooled of four experiments ( n = 3–4 mice per group). RANKL protein expression in LTi cells from WT and LTα −/− mice at d3 SL‐TBI. Data are pooled of three experiments ( n = 3–5 mice per group). The expression of Ltα and Ltβ mRNAs was measured by qPCR in the total thymus from UT WT mice ( n = 4) or at d3 SL‐TBI ( n = 4). MFI of LTα protein in CD45 − and CD45 + thymic cells from UT WT ( n = 6) mice or at d3 SL‐TBI ( n = 6). Representative histogram of LTα (N) and LTβR‐Fc staining (O) in LTi cells from UT WT mice ( n = 6) or at d3 SL‐TBI ( n = 6) or L‐TBI ( n = 6). Data information: Data are shown as mean ± SEM. * P

    Article Snippet: Flow cytometry analysis was performed with a FACSCanto II (BD), and data were analyzed with FlowJo software.

    Techniques: Expressing, Mouse Assay, In Vivo, Isolation, Irradiation, Transplantation Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Staining

    Effect of IL-10 deficiency on CD8 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains or CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days postinfection is shown. (A) Representative flow cytometry plots of cells isolated from brains. (B) Number (top panel) and percentage (bottom panel) of CD3 + brain cells that were CD8 + T cells. (C) Number (top panel) and percentage (bottom panel) of CD3 + cells in CLNs that were CD8 + T cells. The data represent the mean ± SEM from 3 independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on CD8 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains or CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days postinfection is shown. (A) Representative flow cytometry plots of cells isolated from brains. (B) Number (top panel) and percentage (bottom panel) of CD3 + brain cells that were CD8 + T cells. (C) Number (top panel) and percentage (bottom panel) of CD3 + cells in CLNs that were CD8 + T cells. The data represent the mean ± SEM from 3 independent experiments. *, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on Th17 cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. (A to F) Analysis of Il17a (A and B), Il23 (C and D), and Gmcsf (E,F) mRNA expression in the brains of WT (filled squares, solid lines) and IL-10 −/− (open circles, dashed lines) mice. Gene C T values were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A, C, and E) or to uninfected WT mice (B, D, and F) (ΔΔ C T ). Data are pooled from two independent experiments and presented as the mean ± SEM from 6 mice at each time point. (G to K) Flow cytometric analysis of cells isolated from the pooled ( n = 10) brains and CLNs of WT (black bars) and IL-10 −/− (white bars) mice at 7 and 10 days postinfection. (G) Representative flow cytometry plots of Th17 cells, defined as CD3 + CD4 + T cells producing IL-17a. (H to K) Number and percentage of Th17 cells in brain (H and I) and CLN (J and K). The data are plotted as the mean ± SEM from 3 independent experiments.

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on Th17 cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. (A to F) Analysis of Il17a (A and B), Il23 (C and D), and Gmcsf (E,F) mRNA expression in the brains of WT (filled squares, solid lines) and IL-10 −/− (open circles, dashed lines) mice. Gene C T values were normalized to the GAPDH gene, and changes in levels were calculated relative to uninfected mice of the same strain (A, C, and E) or to uninfected WT mice (B, D, and F) (ΔΔ C T ). Data are pooled from two independent experiments and presented as the mean ± SEM from 6 mice at each time point. (G to K) Flow cytometric analysis of cells isolated from the pooled ( n = 10) brains and CLNs of WT (black bars) and IL-10 −/− (white bars) mice at 7 and 10 days postinfection. (G) Representative flow cytometry plots of Th17 cells, defined as CD3 + CD4 + T cells producing IL-17a. (H to K) Number and percentage of Th17 cells in brain (H and I) and CLN (J and K). The data are plotted as the mean ± SEM from 3 independent experiments.

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Expressing, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on CD3 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains and CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days after infection is shown. (A) Representative flow cytometry plots of cells isolated from brain at 7 days after infection. (B and E) Total numbers of cells isolated from brains (B) and CLNs (E). (C and F) Number (C) and percentage (F) of CD3 + T cells isolated from brain. (D and G) Number (D) and percentage (G) of CD3 + T cells isolated from CLNs. The data represent the mean ± SEM from 3 independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on CD3 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains and CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days after infection is shown. (A) Representative flow cytometry plots of cells isolated from brain at 7 days after infection. (B and E) Total numbers of cells isolated from brains (B) and CLNs (E). (C and F) Number (C) and percentage (F) of CD3 + T cells isolated from brain. (D and G) Number (D) and percentage (G) of CD3 + T cells isolated from CLNs. The data represent the mean ± SEM from 3 independent experiments. *, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on regulatory B cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLN of 10 WT (black bars) and IL-10 −/− (white bars) mice at 10 days postinfection is shown. (A) Representative flow cytometry plot of regulatory B cells, defined as CD19 + CD1d hi CD5 + IgM + cells. (B to E) Numbers and percentages of Bregs in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on regulatory B cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLN of 10 WT (black bars) and IL-10 −/− (white bars) mice at 10 days postinfection is shown. (A) Representative flow cytometry plot of regulatory B cells, defined as CD19 + CD1d hi CD5 + IgM + cells. (B to E) Numbers and percentages of Bregs in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on CD19 + B cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10 WT (black bars) and IL-10 −/− (white bars) mice at 10 days postinfection is shown. (A) Representative flow cytometry plot. (B to E) Frequency and percentage of CD19 + B cells in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on CD19 + B cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10 WT (black bars) and IL-10 −/− (white bars) mice at 10 days postinfection is shown. (A) Representative flow cytometry plot. (B to E) Frequency and percentage of CD19 + B cells in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on CD4 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days postinfection (dpi) is shown. (A) Representative flow cytometry plots of cells isolated from brain and CLN at 7 days after infection. (B and C) Number (B) and percentage (C) of CD3 + cells that were CD4 + T cells in brain. (D and E) Number (D) and percentage (E) of CD3 + T cells in CLNs that were CD4 + T cells. The data represent the mean ± SEM from 3 independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on CD4 + T cells in the CNS. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of cells isolated from the pooled brains or CLNs of WT (black bars) or IL-10-deficient (white bars) mice at 5 ( n = 6), 7 ( n = 10), and 10 ( n = 10) days postinfection (dpi) is shown. (A) Representative flow cytometry plots of cells isolated from brain and CLN at 7 days after infection. (B and C) Number (B) and percentage (C) of CD3 + cells that were CD4 + T cells in brain. (D and E) Number (D) and percentage (E) of CD3 + T cells in CLNs that were CD4 + T cells. The data represent the mean ± SEM from 3 independent experiments. *, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    Effect of IL-10 deficiency on ILC2 cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10 WT (black bars) and IL-10 −/− (white bars) mice at 5 days after infection is shown. (A) Representative flow cytometry plots of ILC2 cells defined as IL-7Ra + lineage − Gata3 + . (B to E) Number and percentage of ILC2 cells in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Journal: Journal of Virology

    Article Title: Interleukin-10 Modulation of Virus Clearance and Disease in Mice with Alphaviral Encephalomyelitis

    doi: 10.1128/JVI.01517-17

    Figure Lengend Snippet: Effect of IL-10 deficiency on ILC2 cells. WT and IL-10 −/− B6 mice were intranasally infected with 10 5 PFU TE12. Flow cytometric analysis of isolated cells pooled from the brains and CLNs of 10 WT (black bars) and IL-10 −/− (white bars) mice at 5 days after infection is shown. (A) Representative flow cytometry plots of ILC2 cells defined as IL-7Ra + lineage − Gata3 + . (B to E) Number and percentage of ILC2 cells in brain (B and C) and CLN (D and E). The data are plotted as the mean ± SEM from 3 independent experiments. **, P

    Article Snippet: All flow cytometry data were acquired using the BD FACSCanto II flow cytometer and FACS Diva software and analyzed using FlowJo (v10.3.0).

    Techniques: Mouse Assay, Infection, Flow Cytometry, Isolation, Cytometry

    TLR stimulation causes a progression of Lin + cells from LKS + cells and soluble CD14 augments the acquisition of Mac-1 and F4/80. (A) Sorted LKS + cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin + or Lin - cells are indicated. (B) Sorted LKS + cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS + cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam 3 CSK 4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam 3 CSK 4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures ( * P

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: TLR stimulation causes a progression of Lin + cells from LKS + cells and soluble CD14 augments the acquisition of Mac-1 and F4/80. (A) Sorted LKS + cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin + or Lin - cells are indicated. (B) Sorted LKS + cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS + cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam 3 CSK 4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam 3 CSK 4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures ( * P

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Activation of TLRs through Myd88 bypasses normal differentiation cues and drives monocyte and/or macrophage differentiation of myeloid progenitors. (A) Sorted LKS - cells from C57BL/6 or Myd88 -/- mice were stimulated with medium alone, LPS (10 μg/ml), Pam 3 CSK 4 (1 μg/ml), M-CSF, or GM-CSF. After 72 hr in culture, cells were analyzed by flow cytometry for expression of F4/80. Open histograms depict staining with the isotype matched Ab for F4/80. Percentages given in each histogram indicate the frequencies of F4/80 + cells and the results are representative of three independent experiments. (B) The bar graph depicts yields, i.e., numbers of F4/80 + cell recovered per input progenitor. Data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments ( * P

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Activation of TLRs through Myd88 bypasses normal differentiation cues and drives monocyte and/or macrophage differentiation of myeloid progenitors. (A) Sorted LKS - cells from C57BL/6 or Myd88 -/- mice were stimulated with medium alone, LPS (10 μg/ml), Pam 3 CSK 4 (1 μg/ml), M-CSF, or GM-CSF. After 72 hr in culture, cells were analyzed by flow cytometry for expression of F4/80. Open histograms depict staining with the isotype matched Ab for F4/80. Percentages given in each histogram indicate the frequencies of F4/80 + cells and the results are representative of three independent experiments. (B) The bar graph depicts yields, i.e., numbers of F4/80 + cell recovered per input progenitor. Data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments ( * P

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining

    Lin - stem/progenitor cells express the RP105-MD-1 complex. (A) RP105 or MD-1 were analyzed by flow cytometry on mature peripheral cells or Lin - c-Kit + bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with RP105 or MD-1. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with RP105 or MD-1. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods, and c-Kit was used together with RP105 or MD-1. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) The RP105-MD-1 complex is expressed by hematopoietic progenitor cells. Open histograms depict staining with the isotype matched Abs and the results are representative of two independent experiments.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Lin - stem/progenitor cells express the RP105-MD-1 complex. (A) RP105 or MD-1 were analyzed by flow cytometry on mature peripheral cells or Lin - c-Kit + bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with RP105 or MD-1. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with RP105 or MD-1. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods, and c-Kit was used together with RP105 or MD-1. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) The RP105-MD-1 complex is expressed by hematopoietic progenitor cells. Open histograms depict staining with the isotype matched Abs and the results are representative of two independent experiments.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Staining

    Activation of TLRs through Myd88 on LKS + cells leads to myeloid cell differentiation and stem cell rich Flk-2 - cells enter the cell cycle with TLR ligation. (A) Left, sorted LKS + cells (10,000 cells/well) from C57BL/6 or Myd88 -/- mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Percentages indicate the frequencies of Lin + or Lin - cells. Right, the bar graph depicts yields, i.e., numbers of Lin + cell recovered per input progenitor and the data represent mean values with standard deviations from triplicate cultures ( * P

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Activation of TLRs through Myd88 on LKS + cells leads to myeloid cell differentiation and stem cell rich Flk-2 - cells enter the cell cycle with TLR ligation. (A) Left, sorted LKS + cells (10,000 cells/well) from C57BL/6 or Myd88 -/- mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Percentages indicate the frequencies of Lin + or Lin - cells. Right, the bar graph depicts yields, i.e., numbers of Lin + cell recovered per input progenitor and the data represent mean values with standard deviations from triplicate cultures ( * P

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Activation Assay, Cell Differentiation, Ligation, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Dramatic alterations in B lineage cells, monocytes and/or macrophages and dendritic cells in LPS treated mice. C57BL/6 mice were injected intraperitoneally with 100 μg LPS from E. coli . After 3 or 7 days, bone marrow cells from femurs and tibiae or spleen cells were stained with mAbs to the indicated markers and analyzed by flow cytometry. Percentages of B220 lo AA4.1 + (A), Mac-1 + F4/80 + (B), or Mac-1 + CD11c + (C) cells are indicated. The graphs depict cell numbers of B220 lo AA4.1 + (A), Mac-1 + F4/80 + (B), or Mac-1 + CD11c + (C) cells. Data represent mean values with standard deviations from four mice and are representative of two independent experiments.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Dramatic alterations in B lineage cells, monocytes and/or macrophages and dendritic cells in LPS treated mice. C57BL/6 mice were injected intraperitoneally with 100 μg LPS from E. coli . After 3 or 7 days, bone marrow cells from femurs and tibiae or spleen cells were stained with mAbs to the indicated markers and analyzed by flow cytometry. Percentages of B220 lo AA4.1 + (A), Mac-1 + F4/80 + (B), or Mac-1 + CD11c + (C) cells are indicated. The graphs depict cell numbers of B220 lo AA4.1 + (A), Mac-1 + F4/80 + (B), or Mac-1 + CD11c + (C) cells. Data represent mean values with standard deviations from four mice and are representative of two independent experiments.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry

    TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis. (A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88 -/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19 + cells or Mac-1 + cells (Right) and the data represent mean values with standard deviations from triplicate cultures ( * P

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis. (A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88 -/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19 + cells or Mac-1 + cells (Right) and the data represent mean values with standard deviations from triplicate cultures ( * P

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing

    TLR stimulation drives differentiation of GMPs into F4/80 hi monocytes and/or macrophages. (A) These photomicrographs were prepared with Giemsa-May-Grünwald stained cytocentrifuged slides. (B, C) Sorted GMPs were stimulated with LPS (1 μg/ml), Pam 3 CSK 4 (100 ng/ml), M-CSF, or GM-CSF. After 72 hr in culture, cells were analyzed by flow cytometry for expression of F4/80 and Mac-1. Percentages indicate the frequencies of Mac-1 lo F4/80 lo or Mac-1 hi F4/80 hi cells. CD86, Gr-1, or CD62L were analyzed by flow cytometry on Mac-1 hi F4/80 hi cells. Open histograms depict staining with the appropriate isotype matched Abs. The results are representative of those obtained in two independent experiments. (C) The bar graphs depict cell yields of Mac-1 lo F4/80 lo or Mac-1 hi F4/80 hi cells. The data represent mean values with standard deviations from triplicate cultures and are representative of three independent experiments.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: TLR stimulation drives differentiation of GMPs into F4/80 hi monocytes and/or macrophages. (A) These photomicrographs were prepared with Giemsa-May-Grünwald stained cytocentrifuged slides. (B, C) Sorted GMPs were stimulated with LPS (1 μg/ml), Pam 3 CSK 4 (100 ng/ml), M-CSF, or GM-CSF. After 72 hr in culture, cells were analyzed by flow cytometry for expression of F4/80 and Mac-1. Percentages indicate the frequencies of Mac-1 lo F4/80 lo or Mac-1 hi F4/80 hi cells. CD86, Gr-1, or CD62L were analyzed by flow cytometry on Mac-1 hi F4/80 hi cells. Open histograms depict staining with the appropriate isotype matched Abs. The results are representative of those obtained in two independent experiments. (C) The bar graphs depict cell yields of Mac-1 lo F4/80 lo or Mac-1 hi F4/80 hi cells. The data represent mean values with standard deviations from triplicate cultures and are representative of three independent experiments.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing

    Altered differentiation patterns of single lymphoid progenitors activated via TLRs. Sorted CLPs were cultured in the presence of SCF, FL and IL-7 with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 hr, cultured cells were harvested and washed three times with medium. Single cultured cells were then sorted and re-cultured on OP9 stromal cells in 96-well plates for 10 days in the presence of SCF, FL and IL-7. Positive colonies were examined by flow cytometry (representative examples shown on top row). The frequencies of wells with each of these differentiation patterns are shown along with total numbers of clones observed.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Altered differentiation patterns of single lymphoid progenitors activated via TLRs. Sorted CLPs were cultured in the presence of SCF, FL and IL-7 with medium alone, LPS (10 μg/ml) or Pam 3 CSK 4 (1 μg/ml). After 24 hr, cultured cells were harvested and washed three times with medium. Single cultured cells were then sorted and re-cultured on OP9 stromal cells in 96-well plates for 10 days in the presence of SCF, FL and IL-7. Positive colonies were examined by flow cytometry (representative examples shown on top row). The frequencies of wells with each of these differentiation patterns are shown along with total numbers of clones observed.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Clone Assay

    Lin - c-Kit + progenitors in bone marrow express TLRs and their co-receptors. (A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin - c-Kit + bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: Lin - c-Kit + progenitors in bone marrow express TLRs and their co-receptors. (A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin - c-Kit + bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Staining

    TLR stimulation causes rapid production of F4/80 + cells from LKS - cells. Sorted LKS - cells (10,000 cells/well) from C57BL/6 mice were cultured with LPS (10 μg/ml), Pam 3 CSK 4 (1 μg/ml), M-CSF, or GM-CSF. Virtually no viable cells were recovered from wells with no stimulus and were not studied further. Stimulated wells were analyzed after 24 or 48 hr of culture by flow cytometry for expression of F4/80. Open histograms depict staining with the isotype matched Ab for F4/80. Percentages of F4/80 + cells are representative of three independent experiments.

    Journal: Immunity

    Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

    doi: 10.1016/j.immuni.2006.04.008

    Figure Lengend Snippet: TLR stimulation causes rapid production of F4/80 + cells from LKS - cells. Sorted LKS - cells (10,000 cells/well) from C57BL/6 mice were cultured with LPS (10 μg/ml), Pam 3 CSK 4 (1 μg/ml), M-CSF, or GM-CSF. Virtually no viable cells were recovered from wells with no stimulus and were not studied further. Stimulated wells were analyzed after 24 or 48 hr of culture by flow cytometry for expression of F4/80. Open histograms depict staining with the isotype matched Ab for F4/80. Percentages of F4/80 + cells are representative of three independent experiments.

    Article Snippet: Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).

    Techniques: Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Staining

    Limited T reg cell function in the absence of developmentally established TCR specificities. (A, top) Representative flow cytometric analyses of lymph nodes from mice of the indicated genotypes, gated on CD4 + Foxp3 + cells. (Bottom) EGR2 staining is shown for the indicated cell populations (red, TCRβ − ; black, TCRβ + ; blue, Vα14i + ). (B–D) Lymph node cellularity (B), T cell activation status (C), and splenic T cell cytokine production (D) in Foxp3 CreERT2 Tcra WT/WT (black circles), Foxp3 CreERT2 Tcra WT/Vα14i-StopF (blue circles), Foxp3 CreERT2 Tcra FL/FL (red circles), and Foxp3 CreERT2 Tcra FL/Vα14i-StopF (gray circles) mice. Each circle represents an individual mouse. P-values were calculated using unpaired Student’s t test. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    doi: 10.1084/jem.20161318

    Figure Lengend Snippet: Limited T reg cell function in the absence of developmentally established TCR specificities. (A, top) Representative flow cytometric analyses of lymph nodes from mice of the indicated genotypes, gated on CD4 + Foxp3 + cells. (Bottom) EGR2 staining is shown for the indicated cell populations (red, TCRβ − ; black, TCRβ + ; blue, Vα14i + ). (B–D) Lymph node cellularity (B), T cell activation status (C), and splenic T cell cytokine production (D) in Foxp3 CreERT2 Tcra WT/WT (black circles), Foxp3 CreERT2 Tcra WT/Vα14i-StopF (blue circles), Foxp3 CreERT2 Tcra FL/FL (red circles), and Foxp3 CreERT2 Tcra FL/Vα14i-StopF (gray circles) mice. Each circle represents an individual mouse. P-values were calculated using unpaired Student’s t test. **, P

    Article Snippet: Flow cytometric analysis was performed using an LSRII flow cytometer (BD) and FlowJo software (Tree Star).

    Techniques: Cell Function Assay, Flow Cytometry, Mouse Assay, Staining, Activation Assay

    Preferential accumulation of G113 TCR-expressing T reg cells in skin and draining lymph nodes. (A) Flow cytometric analyses of CD4 + Foxp3 + cells (top left) in the indicated lymph nodes of female G113Tg Foxp3 CreERT2/YFP-Cre Tcra FL/FL mice. Further gating on YFP + cells is shown below. (Right) The percent YFP + Vβ6 + Vα2 + and YFP + TCRβ − cells among total Foxp3 + cells is quantified. (B, left) Representative flow cytometric analyses of CD4 + Foxp3 + cells isolated from tissues of G113Tg Foxp3 CreERT2/YFP-Cre Tcra FL/FL mice. (Right) Quantification of YFP + cells within the total Foxp3 + cell population. (C) 4 × 10 5 Thy1.1 + cells from Foxp3 Thy1.1 mice, 4 × 10 5 Vβ6 + Vα2 + eGFP + cells from tamoxifen-treated G113Tg Foxp3 CreERT2 Tcra FL/FL mice, and 1.6 × 10 6 CD45.1 + eGFP − cells from CD45.1 + Foxp3 GFP mice were transferred into Tcrb −/− / Tcrd −/− mice analyzed 2 wk later. (Left) Representative flow cytometric analysis of Thy1.1 + and Thy1.1 − (Vβ6 + Vα2 + ) T reg cells in the indicated tissues. (Right) The proportion of Thy1.1 − cells (percentage) among total Foxp3 + cells. (D) Representative flow cytometric analyses from an inguinal lymph node of bone marrow chimeric mice. (Right) Plots of the indicated populations of CD45.2 + (red) and CD45.2 − (black) Foxp3 + (left) and Foxp3 − (right) cells, with gates indicating the percent Vβ6 + Vα2 + cells among CD45.2 + Foxp3 + and Foxp3 − populations. (E) Percent CD45.2 + among Foxp3 + cells (left) and numbers of CD45.2 + clusters per imaged section (right) in skin-draining (open circles) or mesenteric/mediastinal (closed circles) lymph nodes. (F) Representative immunofluorescence staining of lymph nodes as indicated. (G) Percent CD8 + (black bars), CD4 + Foxp3 − (green bars), CD4 + Foxp3 + (white bars), and B220 + (blue bars) cells of CD45.2 + cells in skin-draining lymph nodes (total) or in CD45.2 + clusters (clustered) in skin-draining lymph nodes. Error bars represent ± SEM. (H) Representative images showing localization of CD45.1 + (black contours) and CD45.2 + (red dots) Foxp3 + cells in the indicated lymph nodes. (I) Quantification of data in H for both CD4 + Foxp3 + and CD4 + Foxp3 − cells. (A–C) Each circle represents an individual mouse. (A and B) The data are representative of at least two independent experiments with at least two mice of each genotype per experimental group. (C) The data represents the combined results of two experiments with four mice per group each. (D–I) The data are the combined results from the analysis of two independent experiments with a total of three individual mice. (E and I) Each circle represents an imaged section. P-values were calculated using unpaired (A–E) or paired (I) Student’s t test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    doi: 10.1084/jem.20161318

    Figure Lengend Snippet: Preferential accumulation of G113 TCR-expressing T reg cells in skin and draining lymph nodes. (A) Flow cytometric analyses of CD4 + Foxp3 + cells (top left) in the indicated lymph nodes of female G113Tg Foxp3 CreERT2/YFP-Cre Tcra FL/FL mice. Further gating on YFP + cells is shown below. (Right) The percent YFP + Vβ6 + Vα2 + and YFP + TCRβ − cells among total Foxp3 + cells is quantified. (B, left) Representative flow cytometric analyses of CD4 + Foxp3 + cells isolated from tissues of G113Tg Foxp3 CreERT2/YFP-Cre Tcra FL/FL mice. (Right) Quantification of YFP + cells within the total Foxp3 + cell population. (C) 4 × 10 5 Thy1.1 + cells from Foxp3 Thy1.1 mice, 4 × 10 5 Vβ6 + Vα2 + eGFP + cells from tamoxifen-treated G113Tg Foxp3 CreERT2 Tcra FL/FL mice, and 1.6 × 10 6 CD45.1 + eGFP − cells from CD45.1 + Foxp3 GFP mice were transferred into Tcrb −/− / Tcrd −/− mice analyzed 2 wk later. (Left) Representative flow cytometric analysis of Thy1.1 + and Thy1.1 − (Vβ6 + Vα2 + ) T reg cells in the indicated tissues. (Right) The proportion of Thy1.1 − cells (percentage) among total Foxp3 + cells. (D) Representative flow cytometric analyses from an inguinal lymph node of bone marrow chimeric mice. (Right) Plots of the indicated populations of CD45.2 + (red) and CD45.2 − (black) Foxp3 + (left) and Foxp3 − (right) cells, with gates indicating the percent Vβ6 + Vα2 + cells among CD45.2 + Foxp3 + and Foxp3 − populations. (E) Percent CD45.2 + among Foxp3 + cells (left) and numbers of CD45.2 + clusters per imaged section (right) in skin-draining (open circles) or mesenteric/mediastinal (closed circles) lymph nodes. (F) Representative immunofluorescence staining of lymph nodes as indicated. (G) Percent CD8 + (black bars), CD4 + Foxp3 − (green bars), CD4 + Foxp3 + (white bars), and B220 + (blue bars) cells of CD45.2 + cells in skin-draining lymph nodes (total) or in CD45.2 + clusters (clustered) in skin-draining lymph nodes. Error bars represent ± SEM. (H) Representative images showing localization of CD45.1 + (black contours) and CD45.2 + (red dots) Foxp3 + cells in the indicated lymph nodes. (I) Quantification of data in H for both CD4 + Foxp3 + and CD4 + Foxp3 − cells. (A–C) Each circle represents an individual mouse. (A and B) The data are representative of at least two independent experiments with at least two mice of each genotype per experimental group. (C) The data represents the combined results of two experiments with four mice per group each. (D–I) The data are the combined results from the analysis of two independent experiments with a total of three individual mice. (E and I) Each circle represents an imaged section. P-values were calculated using unpaired (A–E) or paired (I) Student’s t test. *, P

    Article Snippet: Flow cytometric analysis was performed using an LSRII flow cytometer (BD) and FlowJo software (Tree Star).

    Techniques: Expressing, Flow Cytometry, Mouse Assay, Isolation, Immunofluorescence, Staining

    Rescue of the polyclonal TCR-dependent gene signature in T reg cells upon expression of the monoclonal G113 TCR. (A) Foxp3 CreERT2 Tcra FL/WT , Foxp3 CreERT2 Tcra FL/FL , G113Tg Foxp3 CreERT2 Tcra WT/WT , and G113Tg Foxp3 CreERT2 Tcra FL/FL mice were treated with tamoxifen on days 0, 1, and 7, and on day 9, the following populations were sorted from pooled spleens and lymph nodes of the indicated mice: CD44 hi CD62L lo and CD44 lo CD62L hi TCRβ + and TCRβ − eGFP + T reg cells from Foxp3 CreERT2 Tcra FL/WT mice; CD44 hi CD62L lo and CD44 lo CD62L hi TCRβ − eGFP + T reg cells from Foxp3 CreERT2 Tcra FL/FL mice; CD44 hi CD62L lo and CD44 lo CD62L hi Vβ6 + eGFP + T reg cells from G113Tg Foxp3 CreERT2 Tcra WT/WT mice; and CD44 hi CD62L lo and CD44 lo CD62L hi Vβ6 + Vα2 + eGFP + T reg cells from G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Strictly TCR-dependent genes were determined as described in the Rescue of the T reg cell TCR-dependent gene signature by monoclonal G113 TCR expression section of Results and discussion and were analyzed for significantly differential expression in the corresponding T reg cell subsets in G113Tg Foxp3 CreERT2 Tcra WT/WT compared with G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Significantly TCR up-regulated genes (red; left) and TCR down-regulated genes (blue; right) are shown for effector (top) and naive (bottom) T reg cell subsets. Genes not significantly up-regulated (left) or down-regulated (right) are shown in black. All genes are shown in gray. Three replicates of each cell subset were generated, with at least five mice pooled for each replicate. A cutoff of 0.05 was set for the obtained p-values, adjusted using Benjamini-Hochberg multiple testing correction, to identify significant gene expression changes for each comparison. (B) Representative flow cytometric analyses of EGR2 expression in naive Vβ6 + Vα2 − (gates and histograms shown in black) and Vβ6 + Vα2 + (gates and histograms shown in red) T reg cells in pooled cervical, axial, brachial, and inguinal (skin draining) lymph nodes (left) and spleens (right) of tamoxifen-treated G113Tg Foxp3 CreERT2 Tcra WT/WT and G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Flow cytometry plots are representative of four independent experiments with at least three mice of each genotype per experiment.

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    doi: 10.1084/jem.20161318

    Figure Lengend Snippet: Rescue of the polyclonal TCR-dependent gene signature in T reg cells upon expression of the monoclonal G113 TCR. (A) Foxp3 CreERT2 Tcra FL/WT , Foxp3 CreERT2 Tcra FL/FL , G113Tg Foxp3 CreERT2 Tcra WT/WT , and G113Tg Foxp3 CreERT2 Tcra FL/FL mice were treated with tamoxifen on days 0, 1, and 7, and on day 9, the following populations were sorted from pooled spleens and lymph nodes of the indicated mice: CD44 hi CD62L lo and CD44 lo CD62L hi TCRβ + and TCRβ − eGFP + T reg cells from Foxp3 CreERT2 Tcra FL/WT mice; CD44 hi CD62L lo and CD44 lo CD62L hi TCRβ − eGFP + T reg cells from Foxp3 CreERT2 Tcra FL/FL mice; CD44 hi CD62L lo and CD44 lo CD62L hi Vβ6 + eGFP + T reg cells from G113Tg Foxp3 CreERT2 Tcra WT/WT mice; and CD44 hi CD62L lo and CD44 lo CD62L hi Vβ6 + Vα2 + eGFP + T reg cells from G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Strictly TCR-dependent genes were determined as described in the Rescue of the T reg cell TCR-dependent gene signature by monoclonal G113 TCR expression section of Results and discussion and were analyzed for significantly differential expression in the corresponding T reg cell subsets in G113Tg Foxp3 CreERT2 Tcra WT/WT compared with G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Significantly TCR up-regulated genes (red; left) and TCR down-regulated genes (blue; right) are shown for effector (top) and naive (bottom) T reg cell subsets. Genes not significantly up-regulated (left) or down-regulated (right) are shown in black. All genes are shown in gray. Three replicates of each cell subset were generated, with at least five mice pooled for each replicate. A cutoff of 0.05 was set for the obtained p-values, adjusted using Benjamini-Hochberg multiple testing correction, to identify significant gene expression changes for each comparison. (B) Representative flow cytometric analyses of EGR2 expression in naive Vβ6 + Vα2 − (gates and histograms shown in black) and Vβ6 + Vα2 + (gates and histograms shown in red) T reg cells in pooled cervical, axial, brachial, and inguinal (skin draining) lymph nodes (left) and spleens (right) of tamoxifen-treated G113Tg Foxp3 CreERT2 Tcra WT/WT and G113Tg Foxp3 CreERT2 Tcra FL/FL mice. Flow cytometry plots are representative of four independent experiments with at least three mice of each genotype per experiment.

    Article Snippet: Flow cytometric analysis was performed using an LSRII flow cytometer (BD) and FlowJo software (Tree Star).

    Techniques: Expressing, Mouse Assay, Generated, Flow Cytometry, Cytometry

    Monoclonal G113 TCR-expressing T reg cells suppress autoimmune disease mediated by a polyclonal effector T cell population. (A–J) Mice of the indicated genotypes were analyzed at 2.5 wk of age. (A and B) Representative flow cytometry plots of TCR Vα and Vβ usage in CD4 + Foxp3 + (A) and CD4 + Foxp3 + TCRβ + (B) lymph node cells in the indicated mice. Further gating of the indicated cellular subsets is indicated by arrows. (C) Representative flow cytometric analysis of lymph node CD4 + Foxp3 + (top) and CD4 + Foxp3 − (bottom) cells in mice of the indicated genotypes. (D) Representative mice of the indicated genotypes. (E) Survival curve for the indicated mice. (F) Representative hematoxylin and eosin staining of tissue sections from mice of the indicated genotypes. (G) Histology scores of tissues from Foxp3 YFP-Cre Tcra WT/FL (black circles), G113Tg Foxp3 YFP-Cre Tcra WT/FL (gray circles), Foxp3 YFP-Cre Tcra FL/FL (red circles), and G113Tg Foxp3 YFP-Cre Tcra FL/FL (blue circles) mice. (H) Percent Foxp3 + cells among CD4 + cells (top left) and percent Ki-67 + (bottom left), percent CD44 hi CD62L lo (top right), and IL-1R2 + (bottom right) cells among Foxp3 + cells in the lymph nodes of mice of the indicated genotypes (as in G). (I) CD4 + Foxp3 − T cell activation and cytokine production in lymph nodes of the indicated mice. (J) The percent Foxp3 + of CD4 + T cells (top) and percent CD44 hi of CD4 + Foxp3 − cells (bottom) in spleens of the indicated mice. Each circle represents an individual mouse. The horizontal bars represent mean value. P-values were calculated using unpaired Student’s t test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    doi: 10.1084/jem.20161318

    Figure Lengend Snippet: Monoclonal G113 TCR-expressing T reg cells suppress autoimmune disease mediated by a polyclonal effector T cell population. (A–J) Mice of the indicated genotypes were analyzed at 2.5 wk of age. (A and B) Representative flow cytometry plots of TCR Vα and Vβ usage in CD4 + Foxp3 + (A) and CD4 + Foxp3 + TCRβ + (B) lymph node cells in the indicated mice. Further gating of the indicated cellular subsets is indicated by arrows. (C) Representative flow cytometric analysis of lymph node CD4 + Foxp3 + (top) and CD4 + Foxp3 − (bottom) cells in mice of the indicated genotypes. (D) Representative mice of the indicated genotypes. (E) Survival curve for the indicated mice. (F) Representative hematoxylin and eosin staining of tissue sections from mice of the indicated genotypes. (G) Histology scores of tissues from Foxp3 YFP-Cre Tcra WT/FL (black circles), G113Tg Foxp3 YFP-Cre Tcra WT/FL (gray circles), Foxp3 YFP-Cre Tcra FL/FL (red circles), and G113Tg Foxp3 YFP-Cre Tcra FL/FL (blue circles) mice. (H) Percent Foxp3 + cells among CD4 + cells (top left) and percent Ki-67 + (bottom left), percent CD44 hi CD62L lo (top right), and IL-1R2 + (bottom right) cells among Foxp3 + cells in the lymph nodes of mice of the indicated genotypes (as in G). (I) CD4 + Foxp3 − T cell activation and cytokine production in lymph nodes of the indicated mice. (J) The percent Foxp3 + of CD4 + T cells (top) and percent CD44 hi of CD4 + Foxp3 − cells (bottom) in spleens of the indicated mice. Each circle represents an individual mouse. The horizontal bars represent mean value. P-values were calculated using unpaired Student’s t test. *, P

    Article Snippet: Flow cytometric analysis was performed using an LSRII flow cytometer (BD) and FlowJo software (Tree Star).

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Staining, Activation Assay

    Inducible replacement of the diverse TCR repertoire with the G113 TCR in mature T reg cells. (A–D) G113Tg Foxp3 CreERT2 Tcra WT/WT and G113Tg Foxp3 CreERT2 Tcra FL/FL mice were treated with tamoxifen on days 0, 3, 7, and 10 and analyzed on day 13. (A) Representative flow cytometric analyses of lymph node CD4 + Foxp3 + (top) and CD4 + Foxp3 − (bottom) cells in G113Tg Foxp3 CreERT2 Tcra WT/WT (left) and G113Tg Foxp3 CreERT2 Tcra FL/FL (right) mice. (B) CD4 + Foxp3 − (left) and CD8 + (right) T cell activation in spleens (top) and lymph nodes (bottom) of G113Tg Foxp3 CreERT2 Tcra WT/WT (open circles) and G113Tg Foxp3 CreERT2 Tcrac FL/FL (closed circles) mice. CD4 + Foxp3 − and CD8 + cells were further gated as either Vβ6 + Vα2 + (G113 + ) or Vβ6 + Vα2 − (G113 − ) cells. (C and D) T cell numbers in lymph nodes (C) and cytokine production in spleens (D) of mice of the indicated genotypes, as in B. (E and F) In vivo suppression assay. (E) Schematic of the experiment. T reg cells were sorted from the following mice on day 0 after tamoxifen administration on days −7 and −6: bulk T reg cells from Foxp3 CreERT2 Tcra WT/WT mice (black circles), Vβ6 + T reg cells from G113Tg Foxp3 CreERT2 Tcra WT/WT mice (gray circles), TCRβ − T reg cells from Foxp3 CreERT2 Tcra FL/FL mice (red circles), and Vβ6 + Vα2 + T reg cells from G113Tg Foxp3 CreERT2 Tcra FL/FL mice (blue circles). CD4 effector T cells were isolated on day 0 from CD45.1 + Foxp3 DTR mice treated with DT on days −5, −4, and −1. On day 0, T reg cells and effector CD4 T cells were transferred into Tcrb −/− Tcrd −/− mice subsequently maintained on weekly doses of DT until analysis on day 28. (F) Numbers of CD45.2 − CD4 + Foxp3 + T reg cells (left) and CD45.1 + CD4 + Foxp3 − cells (middle) and percent IFN-γ + CD45.1 + CD4 + cells (right) in spleens (top) and skin-draining lymph nodes (bottom). Each circle represents an individual mouse. The horizontal bars represent mean value. P-values were calculated using unpaired Student’s t test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    doi: 10.1084/jem.20161318

    Figure Lengend Snippet: Inducible replacement of the diverse TCR repertoire with the G113 TCR in mature T reg cells. (A–D) G113Tg Foxp3 CreERT2 Tcra WT/WT and G113Tg Foxp3 CreERT2 Tcra FL/FL mice were treated with tamoxifen on days 0, 3, 7, and 10 and analyzed on day 13. (A) Representative flow cytometric analyses of lymph node CD4 + Foxp3 + (top) and CD4 + Foxp3 − (bottom) cells in G113Tg Foxp3 CreERT2 Tcra WT/WT (left) and G113Tg Foxp3 CreERT2 Tcra FL/FL (right) mice. (B) CD4 + Foxp3 − (left) and CD8 + (right) T cell activation in spleens (top) and lymph nodes (bottom) of G113Tg Foxp3 CreERT2 Tcra WT/WT (open circles) and G113Tg Foxp3 CreERT2 Tcrac FL/FL (closed circles) mice. CD4 + Foxp3 − and CD8 + cells were further gated as either Vβ6 + Vα2 + (G113 + ) or Vβ6 + Vα2 − (G113 − ) cells. (C and D) T cell numbers in lymph nodes (C) and cytokine production in spleens (D) of mice of the indicated genotypes, as in B. (E and F) In vivo suppression assay. (E) Schematic of the experiment. T reg cells were sorted from the following mice on day 0 after tamoxifen administration on days −7 and −6: bulk T reg cells from Foxp3 CreERT2 Tcra WT/WT mice (black circles), Vβ6 + T reg cells from G113Tg Foxp3 CreERT2 Tcra WT/WT mice (gray circles), TCRβ − T reg cells from Foxp3 CreERT2 Tcra FL/FL mice (red circles), and Vβ6 + Vα2 + T reg cells from G113Tg Foxp3 CreERT2 Tcra FL/FL mice (blue circles). CD4 effector T cells were isolated on day 0 from CD45.1 + Foxp3 DTR mice treated with DT on days −5, −4, and −1. On day 0, T reg cells and effector CD4 T cells were transferred into Tcrb −/− Tcrd −/− mice subsequently maintained on weekly doses of DT until analysis on day 28. (F) Numbers of CD45.2 − CD4 + Foxp3 + T reg cells (left) and CD45.1 + CD4 + Foxp3 − cells (middle) and percent IFN-γ + CD45.1 + CD4 + cells (right) in spleens (top) and skin-draining lymph nodes (bottom). Each circle represents an individual mouse. The horizontal bars represent mean value. P-values were calculated using unpaired Student’s t test. *, P

    Article Snippet: Flow cytometric analysis was performed using an LSRII flow cytometer (BD) and FlowJo software (Tree Star).

    Techniques: Mouse Assay, Flow Cytometry, Activation Assay, In Vivo, Suppression Assay, Isolation

    TCR signaling is normal in Pak2-deficient NKT cells. ( A ) Flow cytometric analysis of TCRβ + Tet + NKT cells from the thymus ( upper panel ) of WT ( Pak2 F/F ; Nur77 -GFP) and KO ( Pak2 F/F ; Cd4 -Cre; Nur77 -GFP) mice divided into their developmental stages by CD44 and NK1.1 expression. ( B ) Flow cytometric analysis of TCR signaling intensity by monitoring GFP signal expressed under the promoter of Nur77 . GFP fluorescence was determined in total TCRβ + Tet + NKT cells and NKT cells between developmental stages 0–III of WT and KO mice. Results are representative of at least two independent experiments for stage 0 NKT cells and five independent experiments for NKT cells at stages I–III.

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: TCR signaling is normal in Pak2-deficient NKT cells. ( A ) Flow cytometric analysis of TCRβ + Tet + NKT cells from the thymus ( upper panel ) of WT ( Pak2 F/F ; Nur77 -GFP) and KO ( Pak2 F/F ; Cd4 -Cre; Nur77 -GFP) mice divided into their developmental stages by CD44 and NK1.1 expression. ( B ) Flow cytometric analysis of TCR signaling intensity by monitoring GFP signal expressed under the promoter of Nur77 . GFP fluorescence was determined in total TCRβ + Tet + NKT cells and NKT cells between developmental stages 0–III of WT and KO mice. Results are representative of at least two independent experiments for stage 0 NKT cells and five independent experiments for NKT cells at stages I–III.

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Flow Cytometry, Mouse Assay, Expressing, Fluorescence

    The developmental defect in NKT cell development is T cell intrinsic. ( A ) Flow cytometric analysis of 1:1 mixed BM chimeras generated by transferring WT ( Pak2 +/+ ; CD45.1 + CD45.2 + ) and KO ( Pak2 F/F ; Cd4 -Cre; CD45.2 + ) donor BM cells containing hematopoietic stem cells (HSCs) into sublethally irradiated C57BL/6 WT hosts that expressed CD45.1. Cells from WT ( upper panel ) and KO ( lower panel ) donor compartments were separated by TCRβ and CD1d/αGC tetramer to gate on NKT cells from the thymus, liver, spleen, and lymph nodes. ( B ) Ratio of the percentage of TCRβ + Tet + NKT cells generated from KO BM cells relative to cells generated from WT BM cells in the thymus, liver, spleen (SPL), and lymph nodes (LN). ( C ) Flow cytometric analysis of TCRβ + Tet + NKT cells from WT and KO donor compartments separated by CD44 and NK1.1 expression from the thymus of chimeric mice ( left panel ). The right panel indicates the ratio of the percentage of TCRβ + Tet + NKT cells generated from KO BM cells relative to cells generated from WT BM cells at different developmental stages [double negative (DN), DP, stages I–III] within the thymus of chimeric mice. ( D ) Flow cytometric analysis of CD44 and NK1.1 expression within TCRβ + Tet + NKT cells from WT and KO donor compartments from the thymus. ( E ) Flow cytometric analysis of CD122 ( upper panel ) and CD127 ( lower panel ) expression within TCRβ + Tet + NKT cells from WT and KO donor compartments from the thymus of chimeric mice. ( F ) Flow cytometric analysis of BrdU incorporation within total TCRβ + Tet + NKT cells from the thymus, liver, spleen, and lymph nodes of chimeric mice. Mice were administered a single i.p. injection of BrdU followed by 3 d BrdU-pulsed water. Graphs in this figure show mean ± SD ( n = 6). Results are representative of at least six independent chimeras. ****0.00001

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: The developmental defect in NKT cell development is T cell intrinsic. ( A ) Flow cytometric analysis of 1:1 mixed BM chimeras generated by transferring WT ( Pak2 +/+ ; CD45.1 + CD45.2 + ) and KO ( Pak2 F/F ; Cd4 -Cre; CD45.2 + ) donor BM cells containing hematopoietic stem cells (HSCs) into sublethally irradiated C57BL/6 WT hosts that expressed CD45.1. Cells from WT ( upper panel ) and KO ( lower panel ) donor compartments were separated by TCRβ and CD1d/αGC tetramer to gate on NKT cells from the thymus, liver, spleen, and lymph nodes. ( B ) Ratio of the percentage of TCRβ + Tet + NKT cells generated from KO BM cells relative to cells generated from WT BM cells in the thymus, liver, spleen (SPL), and lymph nodes (LN). ( C ) Flow cytometric analysis of TCRβ + Tet + NKT cells from WT and KO donor compartments separated by CD44 and NK1.1 expression from the thymus of chimeric mice ( left panel ). The right panel indicates the ratio of the percentage of TCRβ + Tet + NKT cells generated from KO BM cells relative to cells generated from WT BM cells at different developmental stages [double negative (DN), DP, stages I–III] within the thymus of chimeric mice. ( D ) Flow cytometric analysis of CD44 and NK1.1 expression within TCRβ + Tet + NKT cells from WT and KO donor compartments from the thymus. ( E ) Flow cytometric analysis of CD122 ( upper panel ) and CD127 ( lower panel ) expression within TCRβ + Tet + NKT cells from WT and KO donor compartments from the thymus of chimeric mice. ( F ) Flow cytometric analysis of BrdU incorporation within total TCRβ + Tet + NKT cells from the thymus, liver, spleen, and lymph nodes of chimeric mice. Mice were administered a single i.p. injection of BrdU followed by 3 d BrdU-pulsed water. Graphs in this figure show mean ± SD ( n = 6). Results are representative of at least six independent chimeras. ****0.00001

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Flow Cytometry, Generated, Transferring, Irradiation, Expressing, Mouse Assay, BrdU Incorporation Assay, Injection

    Increased NKT cell apoptosis in the absence of Pak2 is independent of Bcl2- or Bcl-xL–dependent survival signals. ( A ) Flow cytometric analysis of TCRβ + Tet + gated NKT cells from the thymus of Pak2 F/F (WT) and Pak2 F/F ; Cd4 -Cre (KO) mice separated by 7-AAD and Annexin V. Total NKT cells were also separated by developmental stage based on CD44 and NK1.1 expression (as described previously). ( B ) Flow cytometric analysis of Bcl-2 and Bcl-xL expression within total thymic NKT cells and NKT cells at stages I–III from WT and Pak2 KO mice. ( C ) Flow cytometric analysis of TCRβ + Tet + gated NKT cells generated within the thymus of Het ( Pak2 F/+ ; Cd4 -Cre ;Bcl2-Tg ) and KO ( Pak2 F/F ; Cd4 -Cre; Bcl2-Tg ) mice overexpressing Bcl2. Heterozygous Pak2 mice that express Bcl2 transgene ( Pak2 F/+ ; Cd4 -Cre ;Bcl2-Tg ) were used as positive controls. TCRβ + Tet + NKT cells were separated into developmental stages I–III by CD44 and NK1.1 expression. ( D ) Flow cytometric analysis of NK1.1, CD44, CD122, and CD127 expression within TCRβ + Tet + NKT cells from the thymus of Het and KO mice overexpressing Bcl2. ( E ) Absolute cell number of stages I–III thymic NKT cells generated in Het and KO mice overexpressing Bcl2. ( F ) Flow cytometric analysis of TCRβ + Tet + NKT cells generated within the liver ( upper panels ), spleen ( middle panels ), and lymph nodes ( lower panels ) of Het and KO mice overexpressing Bcl2. Results are representative of at least three independent experiments. ***0.0001

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: Increased NKT cell apoptosis in the absence of Pak2 is independent of Bcl2- or Bcl-xL–dependent survival signals. ( A ) Flow cytometric analysis of TCRβ + Tet + gated NKT cells from the thymus of Pak2 F/F (WT) and Pak2 F/F ; Cd4 -Cre (KO) mice separated by 7-AAD and Annexin V. Total NKT cells were also separated by developmental stage based on CD44 and NK1.1 expression (as described previously). ( B ) Flow cytometric analysis of Bcl-2 and Bcl-xL expression within total thymic NKT cells and NKT cells at stages I–III from WT and Pak2 KO mice. ( C ) Flow cytometric analysis of TCRβ + Tet + gated NKT cells generated within the thymus of Het ( Pak2 F/+ ; Cd4 -Cre ;Bcl2-Tg ) and KO ( Pak2 F/F ; Cd4 -Cre; Bcl2-Tg ) mice overexpressing Bcl2. Heterozygous Pak2 mice that express Bcl2 transgene ( Pak2 F/+ ; Cd4 -Cre ;Bcl2-Tg ) were used as positive controls. TCRβ + Tet + NKT cells were separated into developmental stages I–III by CD44 and NK1.1 expression. ( D ) Flow cytometric analysis of NK1.1, CD44, CD122, and CD127 expression within TCRβ + Tet + NKT cells from the thymus of Het and KO mice overexpressing Bcl2. ( E ) Absolute cell number of stages I–III thymic NKT cells generated in Het and KO mice overexpressing Bcl2. ( F ) Flow cytometric analysis of TCRβ + Tet + NKT cells generated within the liver ( upper panels ), spleen ( middle panels ), and lymph nodes ( lower panels ) of Het and KO mice overexpressing Bcl2. Results are representative of at least three independent experiments. ***0.0001

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Flow Cytometry, Mouse Assay, Expressing, Generated

    Loss of Pak2 impairs the expression of PLZF and Egr2 in developing NKT cells. ( A ) Flow cytometric analysis of PLZF expression within TCRβ + Tet + NKT cells from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. Left panel shows PLZF expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the PLZF expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. ( B ) Flow cytometric analysis of Egr2 expression within TCRβ + Tet + NKT cells from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. Left panel shows Egr2 expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the Egr2 expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. ( C ) MFI of PLZF from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( D ) MFI of Egr2 from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE ( n = 3). *0.01

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: Loss of Pak2 impairs the expression of PLZF and Egr2 in developing NKT cells. ( A ) Flow cytometric analysis of PLZF expression within TCRβ + Tet + NKT cells from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. Left panel shows PLZF expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the PLZF expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. ( B ) Flow cytometric analysis of Egr2 expression within TCRβ + Tet + NKT cells from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. Left panel shows Egr2 expression in conventional T cells and NKT cells between stages 0 and III in an offset histogram for WT (blue line) and KO (red line) mice. Right panel shows the Egr2 expression overlay for NKT cells at each developmental stage (stages 0–III) from the thymus of WT and KO mice. ( C ) MFI of PLZF from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( D ) MFI of Egr2 from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE ( n = 3). *0.01

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Expressing, Flow Cytometry, Mouse Assay

    Pak2-deficient mice possess fewer NKT1 and NKT2 cells and exhibit reduced IFN-γ– and IL-4–producing capacity. ( A , upper panels ) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and RORγt expression within TCRβ + Tet + gated NKT cells from the thymus of WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. (A, lower panels ) Flow cytometric analysis of NKT2 cells based on PLZF and GATA3 expression within TCRβ + Tet + gated NKT cells from the thymus of WT and Pak2 KO mice. ( B ) Total percentage ( upper panels ) and absolute cell numbers ( lower panels ) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF and RORγt from (A). ( C , upper panels ) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and T-bet expression within TCRβ + Tet + gated NKT cells from the thymus of WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. (C, lower panels ) PLZF hi T-bet low NKT cells were separated by PLZF and RORγt, to distinguish NKT2 (RORγ t − ) and NKT17 (RORγ t + ) cells. ( D ) Total percentage ( upper panels ) and absolute cell numbers ( lower panels ) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF, T-bet, and RORγt from (C). ( E ) Flow cytometric analysis of IFN-γ expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. ( F ) Flow cytometric analysis of IL-4 expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. ( G ) Flow cytometric analysis of IL-17A expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. Graphs in this figure show mean ± SD ( n = 3). Results are representative of at least three independent experiments for transcription factor (A–D) staining and four independent experiments for cytokine staining (E–G). *0.01

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: Pak2-deficient mice possess fewer NKT1 and NKT2 cells and exhibit reduced IFN-γ– and IL-4–producing capacity. ( A , upper panels ) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and RORγt expression within TCRβ + Tet + gated NKT cells from the thymus of WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. (A, lower panels ) Flow cytometric analysis of NKT2 cells based on PLZF and GATA3 expression within TCRβ + Tet + gated NKT cells from the thymus of WT and Pak2 KO mice. ( B ) Total percentage ( upper panels ) and absolute cell numbers ( lower panels ) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF and RORγt from (A). ( C , upper panels ) Flow cytometric analysis of NKT1, NKT2, and NKT17 cells based on PLZF and T-bet expression within TCRβ + Tet + gated NKT cells from the thymus of WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice. (C, lower panels ) PLZF hi T-bet low NKT cells were separated by PLZF and RORγt, to distinguish NKT2 (RORγ t − ) and NKT17 (RORγ t + ) cells. ( D ) Total percentage ( upper panels ) and absolute cell numbers ( lower panels ) of NKT1, NKT2, and NKT17 cells from WT and KO mice based on their expression of PLZF, T-bet, and RORγt from (C). ( E ) Flow cytometric analysis of IFN-γ expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. ( F ) Flow cytometric analysis of IL-4 expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. ( G ) Flow cytometric analysis of IL-17A expression within TCRβ + Tet + gated NKT cells from WT and KO mice after stimulation of total thymocytes with PMA and ionomycin for 4 h. Graphs in this figure show mean ± SD ( n = 3). Results are representative of at least three independent experiments for transcription factor (A–D) staining and four independent experiments for cytokine staining (E–G). *0.01

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Mouse Assay, Flow Cytometry, Expressing, Staining

    Loss of Pak2 in T cells results in a block in the development of NKT cells. ( A ) Flow cytometric analysis of TCRβ + Tet + NKT cells from the thymus, liver, spleen, and lymph nodes from Pak2 F/F (WT) and Pak2 F/F ; Cd4 -Cre (KO) mice. ( B ) Total percentage ( upper panel ) and cell numbers ( lower panel ) of NKT cells (TCRβ + Tet + ) from the thymus, liver, and spleen of WT and KO mice. ( C ) Flow cytometric analysis of CD24, CD44, and NK1.1 expression within total NKT cells (TCRβ + Tet + ) from the thymus of WT and KO mice. To distinguish NKT cell developmental stages, we separated TCRβ + Tet + gated cells by CD44 and NK1.1 ( upper panel ). CD44 − NK1.1 − NKT cells were further separated by side scatter (SSC) and CD24 ( lower panel ). Gating strategies for stages 0, I, II, and III are indicated on the right of each panel. ( D ) Total percentage ( upper panel ) and cell numbers ( lower panel ) of TCRβ + Tet + NKT cells at stages 0–III from WT and KO mice ( upper panel ). ( E ) Flow cytometric analysis of NK1.1 and CD44, CD122 and CD127 expression within TCRβ + Tet + gated NKT cells from the thymus ( upper panel ) and liver ( lower panel ) from WT and KO mice. ( F ) Flow cytometric analysis of CD122 ( upper panel ) and CD127 ( lower panel ) at stages 0–III. Graphs in this figure show mean ± SD ( n = 3). Results are representative of at least three independent experiments. *0.01

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: Loss of Pak2 in T cells results in a block in the development of NKT cells. ( A ) Flow cytometric analysis of TCRβ + Tet + NKT cells from the thymus, liver, spleen, and lymph nodes from Pak2 F/F (WT) and Pak2 F/F ; Cd4 -Cre (KO) mice. ( B ) Total percentage ( upper panel ) and cell numbers ( lower panel ) of NKT cells (TCRβ + Tet + ) from the thymus, liver, and spleen of WT and KO mice. ( C ) Flow cytometric analysis of CD24, CD44, and NK1.1 expression within total NKT cells (TCRβ + Tet + ) from the thymus of WT and KO mice. To distinguish NKT cell developmental stages, we separated TCRβ + Tet + gated cells by CD44 and NK1.1 ( upper panel ). CD44 − NK1.1 − NKT cells were further separated by side scatter (SSC) and CD24 ( lower panel ). Gating strategies for stages 0, I, II, and III are indicated on the right of each panel. ( D ) Total percentage ( upper panel ) and cell numbers ( lower panel ) of TCRβ + Tet + NKT cells at stages 0–III from WT and KO mice ( upper panel ). ( E ) Flow cytometric analysis of NK1.1 and CD44, CD122 and CD127 expression within TCRβ + Tet + gated NKT cells from the thymus ( upper panel ) and liver ( lower panel ) from WT and KO mice. ( F ) Flow cytometric analysis of CD122 ( upper panel ) and CD127 ( lower panel ) at stages 0–III. Graphs in this figure show mean ± SD ( n = 3). Results are representative of at least three independent experiments. *0.01

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Blocking Assay, Flow Cytometry, Mouse Assay, Expressing

    Absence of Pak2 impairs the expression of SLAM6 on developing NKT cells. ( A ) Flow cytometric analysis of side scatter (SSC) and SLAM6 within total NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Cells were gated as SLAM6 − and SLAM6 + . ( B ) MFI of SLAM6 within total NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( C ) Flow cytometric analysis of SLAM6 expression within SLAM6 + NKT cells [gated as in (A)] from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( D ) MFI of SLAM6 within SLAM6 + gated NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE ( n = 3). *0.01

    Journal: The Journal of Immunology Author Choice

    Article Title: Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2

    doi: 10.4049/jimmunol.1501367

    Figure Lengend Snippet: Absence of Pak2 impairs the expression of SLAM6 on developing NKT cells. ( A ) Flow cytometric analysis of side scatter (SSC) and SLAM6 within total NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Cells were gated as SLAM6 − and SLAM6 + . ( B ) MFI of SLAM6 within total NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( C ) Flow cytometric analysis of SLAM6 expression within SLAM6 + NKT cells [gated as in (A)] from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. ( D ) MFI of SLAM6 within SLAM6 + gated NKT cells (TCRβ + Tet + ) from WT ( Pak2 F/F ) and KO ( Pak2 F/F ; Cd4 -Cre) mice at the different NKT cell developmental stages. Results are representative of at least three independent experiments. Graphs in this figure show mean ± SE ( n = 3). *0.01

    Article Snippet: Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility using the Fortessa (BD) flow cytometry system.

    Techniques: Expressing, Flow Cytometry, Mouse Assay

    β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow cytometry. This experiment was performed twice.

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: β 2 M KO HFFFs do not activate KIR2DS1 reporter cells . (A) To control the success of the β 2 M knockout, untreated (WT) HFFFs (black line) and β 2 M KO HFFFs (shaded gray line) were stained with anti-β 2 M, W6/32, and L31 antibodies. Before staining, the cells were stimulated with UV clone (UV) or infected for 72 h with the positive B6 clone (B6 pos) or the negative A8 clone (A8 neg). Unstained cells were included (dotted line). (B) Total lysate of β 2 M KO HFFFs, HFFFs containing CAS9 without sgRNA (CAS9) and WT HFFFs were loaded onto a reduced 10% SDS-PAGE gel. The membrane was blotted with the anti-β 2 M, L31, HC10, anti-M2 Flag, and anti-calnexin (loading control) antibodies. (C) β 2 M KO HFFFs and WT HFFFs were stimulated with UV B6 clone (UV) and infected with the positive B6 clone (B6 pos) or negative A8 clone (A8 neg) for 72 h. An overnight coculture was performed with KIR2DL2 (negative control), KIR2DL1, KIR2DS1, and LILRB1 reporter cells. GFP expression was measured by flow cytometry. This experiment was performed twice.

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Knock-Out, Staining, Infection, SDS Page, Negative Control, Expressing, Flow Cytometry, Cytometry

    Antibody-blocking experiment of reporter cells and HFFFs stimulated with UV B6 clone or infected with the B6 clone . W6/32, 6A4, B1.23.2, and isotype antibodies were added to 72 h UV-stimulated or infected HFFFs, before an overnight coculture with KIR2DS1 and KIR2DL1 reporter cells was performed. GFP expression was measured by flow cytometry. (A) The dot plots are data from a representative experiment. (B) Collection of up to five independent blocking experiments is depicted in bar graphs as the mean ± SD of individual samples with * p

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: Antibody-blocking experiment of reporter cells and HFFFs stimulated with UV B6 clone or infected with the B6 clone . W6/32, 6A4, B1.23.2, and isotype antibodies were added to 72 h UV-stimulated or infected HFFFs, before an overnight coculture with KIR2DS1 and KIR2DL1 reporter cells was performed. GFP expression was measured by flow cytometry. (A) The dot plots are data from a representative experiment. (B) Collection of up to five independent blocking experiments is depicted in bar graphs as the mean ± SD of individual samples with * p

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Blocking Assay, Infection, Expressing, Flow Cytometry, Cytometry

    KIR2DS1 reporter cells are not activated by conventional human leukocyte antigen (HLA) class I molecules . (A) The 721.221-HLA transfectants were stained with W6/32 (black line), and measured by flow cytometry. IgG2a antibody was used as isotype control (dotted line). (B) Dot plots of a selection of representative data from the same experiment. A coculture of the indicated reporter cells together with 721.221 cells containing HLA-C07:01 (221 Cw7, C1) or HLA-C06:02 (221-Cw6, C2) is shown. GFP expression was determined by flow cytometry. The E:T ratio was 1:3. (C) The data are depicted as the mean ± SD of individual samples collected from five independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: KIR2DS1 reporter cells are not activated by conventional human leukocyte antigen (HLA) class I molecules . (A) The 721.221-HLA transfectants were stained with W6/32 (black line), and measured by flow cytometry. IgG2a antibody was used as isotype control (dotted line). (B) Dot plots of a selection of representative data from the same experiment. A coculture of the indicated reporter cells together with 721.221 cells containing HLA-C07:01 (221 Cw7, C1) or HLA-C06:02 (221-Cw6, C2) is shown. GFP expression was determined by flow cytometry. The E:T ratio was 1:3. (C) The data are depicted as the mean ± SD of individual samples collected from five independent experiments.

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Selection, Expressing

    KIR2DS1 reporter cells are not activated by interferon (IFN)-γ stimulated human fetal foreskin and primary dermal fibroblasts of healthy individuals . (A) Total human leukocyte antigen (HLA) class I (W6/32) and HLA-C/-E (DT9) expression levels of untreated and IFN-γ stimulated HFFFs (C2/C2) and DFs of donor CMV307 (C1/C1) and CMV0005 (C1/C2) were measured by flow cytometry. Gray = untreated, black = IFN-γ stimulated (500 U/ml, 72 h) and dotted line = isotype control. (B) The untreated and IFN-γ-stimulated fibroblasts were cocultured overnight with the indicated reporter cells, and green fluorescent protein (GFP) expression was measured by flow cytometry. Three independent experiments are depicted in the bar graphs as the mean ± SD of individual samples.

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: KIR2DS1 reporter cells are not activated by interferon (IFN)-γ stimulated human fetal foreskin and primary dermal fibroblasts of healthy individuals . (A) Total human leukocyte antigen (HLA) class I (W6/32) and HLA-C/-E (DT9) expression levels of untreated and IFN-γ stimulated HFFFs (C2/C2) and DFs of donor CMV307 (C1/C1) and CMV0005 (C1/C2) were measured by flow cytometry. Gray = untreated, black = IFN-γ stimulated (500 U/ml, 72 h) and dotted line = isotype control. (B) The untreated and IFN-γ-stimulated fibroblasts were cocultured overnight with the indicated reporter cells, and green fluorescent protein (GFP) expression was measured by flow cytometry. Three independent experiments are depicted in the bar graphs as the mean ± SD of individual samples.

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry

    The KIR2DS1 reporter cell recognizes a ligand on human fetal foreskin fibroblasts (HFFFs) infected with specific human cytomegalovirus (HCMV) strains . (A) Uninfected HFFFs and 24, 48, and 72 h infected HFFFs with HCMV TB40/E strain with an MOI of 10 were cocultured overnight with the reporter cells as indicated. A representative experiment of two independent time course cocultures is shown. (B) HFFFs were infected with six different TB40/E-isolated clones (E1, E4, F5, G1, D6, and F8) in 10-fold serial dilutions for 72 h and cocultured with KIR2DS1 reporter cells. A representative experiment from over three independent experiments is depicted. (C) The infectivities of three positive and three negative TB40/E clones were calculated by TCID 50 assay and are depicted in the table (virus titers in PFU/ml). Viral particles were isolated from the supernatant, lysed, and loaded onto a 12% SDS-PAGE gel for western blotting with anti-pp28 antibody. Positive and negative clones were paired based on approximately the same infectivity. Equal amounts of lysate from these pairs were loaded onto the gel. (−) indicates a negative clone, (+) positive clone. (D) HFFFs were stimulated with UV-inactivated TB40/E, infected with the positive B6 or negative A8 clone for 72 h. Forty-eight hours p.i. KIR2DL2 (negative control), KIR2DL1, LILRB1 (positive control), and KIR2DS1 reporter cells were added. After an overnight coculture, the GFP expression was measured using flow cytometry. The data are depicted as the mean ± SD of individual samples collected from five independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: The KIR2DS1 reporter cell recognizes a ligand on human fetal foreskin fibroblasts (HFFFs) infected with specific human cytomegalovirus (HCMV) strains . (A) Uninfected HFFFs and 24, 48, and 72 h infected HFFFs with HCMV TB40/E strain with an MOI of 10 were cocultured overnight with the reporter cells as indicated. A representative experiment of two independent time course cocultures is shown. (B) HFFFs were infected with six different TB40/E-isolated clones (E1, E4, F5, G1, D6, and F8) in 10-fold serial dilutions for 72 h and cocultured with KIR2DS1 reporter cells. A representative experiment from over three independent experiments is depicted. (C) The infectivities of three positive and three negative TB40/E clones were calculated by TCID 50 assay and are depicted in the table (virus titers in PFU/ml). Viral particles were isolated from the supernatant, lysed, and loaded onto a 12% SDS-PAGE gel for western blotting with anti-pp28 antibody. Positive and negative clones were paired based on approximately the same infectivity. Equal amounts of lysate from these pairs were loaded onto the gel. (−) indicates a negative clone, (+) positive clone. (D) HFFFs were stimulated with UV-inactivated TB40/E, infected with the positive B6 or negative A8 clone for 72 h. Forty-eight hours p.i. KIR2DL2 (negative control), KIR2DL1, LILRB1 (positive control), and KIR2DS1 reporter cells were added. After an overnight coculture, the GFP expression was measured using flow cytometry. The data are depicted as the mean ± SD of individual samples collected from five independent experiments. * p

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Infection, Isolation, Clone Assay, SDS Page, Western Blot, Negative Control, Positive Control, Expressing, Flow Cytometry, Cytometry

    2B4 reporter system for activating killer cell Ig-like receptor (KIR) . (A) Cell-surface expression on the indicated reporter cells was measured by flow cytometry after staining the cells with PAN2D (clone NKVFS1, recognizes all KIRs) or anti-HA antibody (black line, left panel). Parental 2B4 cells containing only the adaptor protein were used as a negative control. To test the function of these reporter cells, plate-bound PAN2D (clone NKVFS1) or anti-HA (LILRB1 reporter only) antibody crosslinking (black line) was used to engage KIR or LILRB1 molecules on the reporter cells during an overnight incubation (right panel). IgG1 antibody was used as isotype control (dotted line). (B) Table depicting amino acid positions of polymorphic sites in the extracellular domain of KIR2DL1 and -2DS1 alleles. Large letters indicate amino acids present in the majority of alleles, and small letters indicate amino acids that are present in few alleles. At position 70, a unique amino acid in KIR2DL1 and -2DS1, threonine (T) and lysine (K), respectively, was found. Allele sequences were aligned using the alignment tool in the IPD-KIR database . (C) KIR2DS1 (K70T) was transfected into the 2B4 reporter system and an overnight coculture was performed using KIR2DL1, -2DS1, or -2DS1 (K70T) reporter cells together with 721.221 cells expressing human leukocyte antigen (HLA)-Cw3 (C1) or HLA-Cw6 (C2). The next day green fluorescent protein (GFP) expression was measured by flow cytometry. The E:T ratio of the coculture was 1:3.

    Journal: Frontiers in Immunology

    Article Title: Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

    doi: 10.3389/fimmu.2017.00298

    Figure Lengend Snippet: 2B4 reporter system for activating killer cell Ig-like receptor (KIR) . (A) Cell-surface expression on the indicated reporter cells was measured by flow cytometry after staining the cells with PAN2D (clone NKVFS1, recognizes all KIRs) or anti-HA antibody (black line, left panel). Parental 2B4 cells containing only the adaptor protein were used as a negative control. To test the function of these reporter cells, plate-bound PAN2D (clone NKVFS1) or anti-HA (LILRB1 reporter only) antibody crosslinking (black line) was used to engage KIR or LILRB1 molecules on the reporter cells during an overnight incubation (right panel). IgG1 antibody was used as isotype control (dotted line). (B) Table depicting amino acid positions of polymorphic sites in the extracellular domain of KIR2DL1 and -2DS1 alleles. Large letters indicate amino acids present in the majority of alleles, and small letters indicate amino acids that are present in few alleles. At position 70, a unique amino acid in KIR2DL1 and -2DS1, threonine (T) and lysine (K), respectively, was found. Allele sequences were aligned using the alignment tool in the IPD-KIR database . (C) KIR2DS1 (K70T) was transfected into the 2B4 reporter system and an overnight coculture was performed using KIR2DL1, -2DS1, or -2DS1 (K70T) reporter cells together with 721.221 cells expressing human leukocyte antigen (HLA)-Cw3 (C1) or HLA-Cw6 (C2). The next day green fluorescent protein (GFP) expression was measured by flow cytometry. The E:T ratio of the coculture was 1:3.

    Article Snippet: Flow cytometry data were analyzed using FlowJo software (TreeStar, OR, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Negative Control, Incubation, Transfection

    hTERT expression in CSCs is mutually exclusive with the mesenchymal phenotype. (A) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) showing that the loss of mesenchymal phenotype in hTERT high CSCs mediated by PD173074 is associated with the loss of hTERT expression. However, acquisition of a mesenchymal phenotype in hTERT -/low CSCs mediated by TGF-β is associated with increased hTERT expression. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Flow cytometry overlay histogram analysis of N-cadherin, E-cadherin and hTERT showing that hTERT high CSCs treated with PD173074 lose their mesenchymal phenotype, which was associated with a loss of hTERT expression. Additionally, hTERT -/low CSCs treated with TGF-β acquired a mesenchymal phenotype, which was associated with increased hTERT expression. For comparison, an isotype control was used to define the positive and negative population for each marker.

    Journal: Biology Open

    Article Title: Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells

    doi: 10.1242/bio.034181

    Figure Lengend Snippet: hTERT expression in CSCs is mutually exclusive with the mesenchymal phenotype. (A) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) showing that the loss of mesenchymal phenotype in hTERT high CSCs mediated by PD173074 is associated with the loss of hTERT expression. However, acquisition of a mesenchymal phenotype in hTERT -/low CSCs mediated by TGF-β is associated with increased hTERT expression. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Flow cytometry overlay histogram analysis of N-cadherin, E-cadherin and hTERT showing that hTERT high CSCs treated with PD173074 lose their mesenchymal phenotype, which was associated with a loss of hTERT expression. Additionally, hTERT -/low CSCs treated with TGF-β acquired a mesenchymal phenotype, which was associated with increased hTERT expression. For comparison, an isotype control was used to define the positive and negative population for each marker.

    Article Snippet: Immediately before flow cytometry analysis, 2 µg/ml propidium iodide (Sigma-Aldrich) was added to exclude dead cells.

    Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Marker

    Verification of hTERT knockdown and overexpression in breast CSCs. (A) Flow cytometry plot for cell surface markers CD44 and CD24 in sorted CSCs confirming pure population. Gating is set to an isotype control. (B) Western blot analysis confirming hTERT knockdown and upregulation compared with control scrambled cells. β-actin was used to ensure the loading of equal amounts of protein. (C) Real-time qRT-PCR analysis of hTERT mRNA expression confirming downregulation and upregulation compared with control scrambled cells. β-Actin mRNA was used to normalize the variability in template loading. The data are reported as the means±s.d. (D) Confocal immunofluorescence images for hTERT (green) confirming hTERT knockdown. Nuclei were stained with DAPI (blue). Scale bars: 60 µM.

    Journal: Biology Open

    Article Title: Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells

    doi: 10.1242/bio.034181

    Figure Lengend Snippet: Verification of hTERT knockdown and overexpression in breast CSCs. (A) Flow cytometry plot for cell surface markers CD44 and CD24 in sorted CSCs confirming pure population. Gating is set to an isotype control. (B) Western blot analysis confirming hTERT knockdown and upregulation compared with control scrambled cells. β-actin was used to ensure the loading of equal amounts of protein. (C) Real-time qRT-PCR analysis of hTERT mRNA expression confirming downregulation and upregulation compared with control scrambled cells. β-Actin mRNA was used to normalize the variability in template loading. The data are reported as the means±s.d. (D) Confocal immunofluorescence images for hTERT (green) confirming hTERT knockdown. Nuclei were stained with DAPI (blue). Scale bars: 60 µM.

    Article Snippet: Immediately before flow cytometry analysis, 2 µg/ml propidium iodide (Sigma-Aldrich) was added to exclude dead cells.

    Techniques: Over Expression, Flow Cytometry, Cytometry, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

    hTERT defines CSC properties. (A) Confocal immunofluorescence images for ALDH1A1 (green) showing that hTERT high CSCs are positive for ALDH1A1, whereas hTERT -/low CSCs do not express ALDH1A1. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Confocal immunofluorescence images for β-catenin (green) showing cytoplasmic localization of β-catenin in hTERT -/low CSCs, whereas hTERT high CSCs showed nuclear localization of β-catenin. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (C) Side population (SP) analysis by flow cytometry indicating more SP cells in hTERT high CSCs than in hTERT -/low CSCs, which have fewer SP cells than do control CSCs. (D) Flow cytometry analysis of CD133 showing that hTERT high CSCs have higher CD133 expression than do control CSCs, whereas hTERT -/low CSCs are negative for CD133 expression. An isotype control was used to define the positive and negative populations. (E–F) hTERT high CSCs, hTERT -/low CSCs and control CSCs were exposed to increasing concentrations of cisplatin (E) or doxorubicin (F) for 24 h. Cell viability was determined by Annexin-V-FITC and PI apoptosis detection kits. hTERT high CSCs showed more significant resistance to cisplatin and doxorubicin than did control CSCs, whereas hTERT -/low CSCs exhibited a relative loss of chemoresistance capabilities ( P

    Journal: Biology Open

    Article Title: Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells

    doi: 10.1242/bio.034181

    Figure Lengend Snippet: hTERT defines CSC properties. (A) Confocal immunofluorescence images for ALDH1A1 (green) showing that hTERT high CSCs are positive for ALDH1A1, whereas hTERT -/low CSCs do not express ALDH1A1. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (B) Confocal immunofluorescence images for β-catenin (green) showing cytoplasmic localization of β-catenin in hTERT -/low CSCs, whereas hTERT high CSCs showed nuclear localization of β-catenin. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (C) Side population (SP) analysis by flow cytometry indicating more SP cells in hTERT high CSCs than in hTERT -/low CSCs, which have fewer SP cells than do control CSCs. (D) Flow cytometry analysis of CD133 showing that hTERT high CSCs have higher CD133 expression than do control CSCs, whereas hTERT -/low CSCs are negative for CD133 expression. An isotype control was used to define the positive and negative populations. (E–F) hTERT high CSCs, hTERT -/low CSCs and control CSCs were exposed to increasing concentrations of cisplatin (E) or doxorubicin (F) for 24 h. Cell viability was determined by Annexin-V-FITC and PI apoptosis detection kits. hTERT high CSCs showed more significant resistance to cisplatin and doxorubicin than did control CSCs, whereas hTERT -/low CSCs exhibited a relative loss of chemoresistance capabilities ( P

    Article Snippet: Immediately before flow cytometry analysis, 2 µg/ml propidium iodide (Sigma-Aldrich) was added to exclude dead cells.

    Techniques: Immunofluorescence, Staining, Flow Cytometry, Cytometry, Expressing

    hTERT plays a critical role regulating the epithelial-to-mesenchymal transition in CSCs. (A) Phase-contrast images showing hTERT -/low CSCs have an epithelial phenotype, whereas hTERT high CSCs have a mesenchymal phenotype. (B) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) in control CSCs, CSCs overexpressing hTERT and hTERT-knockdown CSCs. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (C) The expression levels of mRNAs encoding N-cadherin, Zeb1, Snail, Slug, Twist, Vimentin and Desmoplakin in CSCs overexpressing hTERT and hTERT-knockdown CSCs relative to control CSCs as determined by real-time qRT-PCR. The data are reported as the means±s.d. (D) Flow cytometry overlay histogram analysis of N-cadherin and E-cadherin in control CSCs and CSCs overexpressing hTERT and hTERT-knockdown CSCs. For comparison, an isotype control was used to define the positive and negative population for each marker.

    Journal: Biology Open

    Article Title: Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells

    doi: 10.1242/bio.034181

    Figure Lengend Snippet: hTERT plays a critical role regulating the epithelial-to-mesenchymal transition in CSCs. (A) Phase-contrast images showing hTERT -/low CSCs have an epithelial phenotype, whereas hTERT high CSCs have a mesenchymal phenotype. (B) Confocal immunofluorescence images for N-cadherin (green), E-cadherin (red) and Snail+Slug (green) in control CSCs, CSCs overexpressing hTERT and hTERT-knockdown CSCs. Nuclei were stained with DAPI (blue). Scale bars: 60 µM. (C) The expression levels of mRNAs encoding N-cadherin, Zeb1, Snail, Slug, Twist, Vimentin and Desmoplakin in CSCs overexpressing hTERT and hTERT-knockdown CSCs relative to control CSCs as determined by real-time qRT-PCR. The data are reported as the means±s.d. (D) Flow cytometry overlay histogram analysis of N-cadherin and E-cadherin in control CSCs and CSCs overexpressing hTERT and hTERT-knockdown CSCs. For comparison, an isotype control was used to define the positive and negative population for each marker.

    Article Snippet: Immediately before flow cytometry analysis, 2 µg/ml propidium iodide (Sigma-Aldrich) was added to exclude dead cells.

    Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Marker

    The autoimmune phenotype of Roquin san/san mice requires T cell activation through CD28, and T FH cells are expanded cell autonomously. (a, left) Detection of IgG-ANA using Hep-2 slides in sera from 8-wk-old female Roquin san/san and Roquin san/san Cd28 −/− mice ( n = 5 per group). (right) Detection of anti-dsDNA IgG serum antibodies in 6-mo-old female Roquin san/san and Roquin san/san Cd28 −/− mice determined by staining C. luciliae slides. Data are representative of three independent experiments ( n ≥ 5 per group). (b) Score of nephritis severity in 6-mo-old female Roquin +/+ Roquin san/san and Roquin san/san Cd28 −/− mice as determined by histological analysis defined by the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Each symbol represents one mouse. Horizontal bars indicate medians. (c) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right). Histology from Roquin san/san Cd28 −/− animals was much less severe, with normal H E appearances and few electron-dense deposits (arrows) in the mesangium. Bars: (left) 100 µm; (right) 10 µm. (d) Representative flow cytometric contour plots of CD4 + CXCR5 + PD-1 high T FH cells in 10-wk-old Roquin san/san mice and control littermates. Data are representative of five independent experiments ( n = 4 per group), and the numbers in the plots represent percentages. (e) Dot plots representing percentages of CD4 + CXCR5 + PD-1 high T FH cells from SRBC-immunized chimeric mice generated by reconstituting sublethally irradiated mice with a 1:1 mix of either Roquin +/+ .Ly5 a / Roquin +/+ .Ly5 b (left) or Roquin +/+ .Ly5 a / Roquin san/san .Ly5 b (right). Data are representative of three independent experiments ( n = 4 per group). Each symbol represents the Ly5 a or Ly5 b population derived from one mouse. (f) ELISA was used to determine culture supernatant IL-21 levels from 24-h splenocyte cultures from Roquin san/san mice and littermate controls in the presence of PMA and ionomycin. Error bars indicate means ± SEM. Data are representative of three independent tests where each sample was run in triplicate.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: The autoimmune phenotype of Roquin san/san mice requires T cell activation through CD28, and T FH cells are expanded cell autonomously. (a, left) Detection of IgG-ANA using Hep-2 slides in sera from 8-wk-old female Roquin san/san and Roquin san/san Cd28 −/− mice ( n = 5 per group). (right) Detection of anti-dsDNA IgG serum antibodies in 6-mo-old female Roquin san/san and Roquin san/san Cd28 −/− mice determined by staining C. luciliae slides. Data are representative of three independent experiments ( n ≥ 5 per group). (b) Score of nephritis severity in 6-mo-old female Roquin +/+ Roquin san/san and Roquin san/san Cd28 −/− mice as determined by histological analysis defined by the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Each symbol represents one mouse. Horizontal bars indicate medians. (c) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right). Histology from Roquin san/san Cd28 −/− animals was much less severe, with normal H E appearances and few electron-dense deposits (arrows) in the mesangium. Bars: (left) 100 µm; (right) 10 µm. (d) Representative flow cytometric contour plots of CD4 + CXCR5 + PD-1 high T FH cells in 10-wk-old Roquin san/san mice and control littermates. Data are representative of five independent experiments ( n = 4 per group), and the numbers in the plots represent percentages. (e) Dot plots representing percentages of CD4 + CXCR5 + PD-1 high T FH cells from SRBC-immunized chimeric mice generated by reconstituting sublethally irradiated mice with a 1:1 mix of either Roquin +/+ .Ly5 a / Roquin +/+ .Ly5 b (left) or Roquin +/+ .Ly5 a / Roquin san/san .Ly5 b (right). Data are representative of three independent experiments ( n = 4 per group). Each symbol represents the Ly5 a or Ly5 b population derived from one mouse. (f) ELISA was used to determine culture supernatant IL-21 levels from 24-h splenocyte cultures from Roquin san/san mice and littermate controls in the presence of PMA and ionomycin. Error bars indicate means ± SEM. Data are representative of three independent tests where each sample was run in triplicate.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Activation Assay, Staining, Microscopy, Flow Cytometry, Generated, Irradiation, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Lack of IL-21 does not affect the phenotype, T FH cell accumulation, or GC formation of Roquin san/san mice. (a) IgG ANAs in the serum of mice of the genotypes indicated, detected by immunofluorescence using Hep-2 substrate. Data are representative of three independent experiments ( n ≥ 5 mice per group). (b) Score of ANA staining intensity by confocal microscopy from sera taken from mice of the indicated genotypes. Data are representative of three independent experiments ( n ≥ 5 mice per group). (c) Basal serum total IgG, IgG1, and IgE measured by ELISA. Data are representative of two independent experiments ( n = 5 mice per group). (d) Lymph node and spleen weight in grams for mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 per group). (e) Flow cytometric contour plots and dot plots of PD-1 high CXCR5 + CD4 + T FH cells and (f) GL-7 + CD95 + B220 + GC cells from mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 mice per group). In e and f, the numbers in the plots represent percentages.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: Lack of IL-21 does not affect the phenotype, T FH cell accumulation, or GC formation of Roquin san/san mice. (a) IgG ANAs in the serum of mice of the genotypes indicated, detected by immunofluorescence using Hep-2 substrate. Data are representative of three independent experiments ( n ≥ 5 mice per group). (b) Score of ANA staining intensity by confocal microscopy from sera taken from mice of the indicated genotypes. Data are representative of three independent experiments ( n ≥ 5 mice per group). (c) Basal serum total IgG, IgG1, and IgE measured by ELISA. Data are representative of two independent experiments ( n = 5 mice per group). (d) Lymph node and spleen weight in grams for mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 per group). (e) Flow cytometric contour plots and dot plots of PD-1 high CXCR5 + CD4 + T FH cells and (f) GL-7 + CD95 + B220 + GC cells from mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 5 mice per group). In e and f, the numbers in the plots represent percentages.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Immunofluorescence, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    Spontaneous GC and T FH formation are corrected by loss of SAP in Roquin san/san mice. (a) CD4 + CXCR5 + PD-1 high T FH cells in unimmunized 10-wk-old Roquin san/san and Roquin san/san Sap −/− mice (P = 0.0056). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (b) B220 + GL-7 + CD95 + GC B cells in unimmunized 10-wk-old Roquin san/san Sap +/+ and Roquin san/san Sap −/− mice (P = 0.0007). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (c) Photomicrographs of frozen spleen sections from unimmunized 6-mo-old mice of the indicated genotypes stained with IgD (brown; all panels), PNA (blue; left), TCRβ (blue; middle), and PD-1 (blue; right). Bars, 200 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: Spontaneous GC and T FH formation are corrected by loss of SAP in Roquin san/san mice. (a) CD4 + CXCR5 + PD-1 high T FH cells in unimmunized 10-wk-old Roquin san/san and Roquin san/san Sap −/− mice (P = 0.0056). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (b) B220 + GL-7 + CD95 + GC B cells in unimmunized 10-wk-old Roquin san/san Sap +/+ and Roquin san/san Sap −/− mice (P = 0.0007). Representative flow cytometric contour plots are shown (right). Data are representative of four independent experiments ( n = 4 per group). (c) Photomicrographs of frozen spleen sections from unimmunized 6-mo-old mice of the indicated genotypes stained with IgD (brown; all panels), PNA (blue; left), TCRβ (blue; middle), and PD-1 (blue; right). Bars, 200 µm.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Flow Cytometry, Staining

    Th1 and Th2 cells are present in Roquin san/san mice in the absence of SAP and T FH cells, but not non-T FH effector cells, induce a GC response in wild-type mice. (a) Representative flow cytometric contour plots and (b) graphical analysis of GATA3 + CD44 high CD4 + and Tbet + CD44 high CD4 + cells in mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 4 per group). (c) Representative dot plots of lymph node CD4 + PD-1 high CXCR5 + (left), Tbet + CD44 high CD4 + (middle), and GATA3 + CD44 high CD4 + (right) cells. (d) Experimental outline for adoptive transfer of Roquin san/san CD4 + CD45.2 CD44 high PD-1 high CXCR5 + or CD4 + CD45.2 CD44 high PD-1 − CXCR5 − T cells into CD45.1 C57BL/6 mice. (e) Flow cytometric contour plots and (f) dot plots of B220 + GL-7 + CD95 + GC B cells from CD45.1 C57BL/6 recipients 3 wk after adoptive transfer of the indicated cell type. Data were generated from three mice per group (**, P > 0.001). In a, d, and e, the numbers in the plots represent percentages.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: Th1 and Th2 cells are present in Roquin san/san mice in the absence of SAP and T FH cells, but not non-T FH effector cells, induce a GC response in wild-type mice. (a) Representative flow cytometric contour plots and (b) graphical analysis of GATA3 + CD44 high CD4 + and Tbet + CD44 high CD4 + cells in mice of the indicated genotypes. Data are representative of two independent experiments ( n ≥ 4 per group). (c) Representative dot plots of lymph node CD4 + PD-1 high CXCR5 + (left), Tbet + CD44 high CD4 + (middle), and GATA3 + CD44 high CD4 + (right) cells. (d) Experimental outline for adoptive transfer of Roquin san/san CD4 + CD45.2 CD44 high PD-1 high CXCR5 + or CD4 + CD45.2 CD44 high PD-1 − CXCR5 − T cells into CD45.1 C57BL/6 mice. (e) Flow cytometric contour plots and (f) dot plots of B220 + GL-7 + CD95 + GC B cells from CD45.1 C57BL/6 recipients 3 wk after adoptive transfer of the indicated cell type. Data were generated from three mice per group (**, P > 0.001). In a, d, and e, the numbers in the plots represent percentages.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Flow Cytometry, Adoptive Transfer Assay, Generated

    Heterozygosity for Bcl6 reduces the magnitude of the GC response in Roquin +/+ and Roquin san/san mice and ameliorates the lupus-like phenotype of Roquin san/san mice. (a) Flow cytometric contour plots (left) and graphical analysis (right) of B220 + GL-7 + CD95 + GC B cells in 10-wk-old wild-type ( Bcl6 +/+ ) and Bcl6 +/− mice 8 d after SRBC immunization (P = 0.0011). Data are representative of four independent experiments ( n = 4 per group). (b) Flow cytometric contour plots (left) and dot plots (right) showing B220 + GL-7 + CD95 + GC B cells from 10-wk-old naive Roquin san/san Bcl6 +/+ and Roquin san/san Bcl6 +/− mice. Data are representative of five independent experiments ( n ≥ 4 per group). (c) Representative determination of serum IgG anti-dsDNA from 6-mo-old female Roquin +/+ Bcl6 +/+ , Roquin san/san Bcl6 +/+ , and Roquin san/san Bcl6 +/− mice, determined by immunofluorescence staining of C. luciliae substrate. Data shown reflect the occurrence ( n ≥ 6 mice per group); three out of six Roquin san/san Bcl6 +/− mice had low intensity staining (illustrated in the fourth panel from left), and three out of six were negative (illustrated in the third panel from left). (d) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right) from 6-mo-old mice of the indicated genotypes. Roquin san/san animals show widespread mesangial proliferative lesions with moderate interstitial infiltrate. There are multiple electron-dense deposits (arrows). Histological changes in Roquin san/san Bcl6 +/− mice were mild, with occasional electron-dense deposits visible on electron microscopy in two individuals (far right). Images are representative ( n ≥ 4 per group). Bars: (H E) 100 µm in all panels; (electron microscopy) 5 µm in the Roquin +/+ Bcl6 +/+ and Roquin san/san Bcl6 +/+ panels, 2 µm in the left Roquin san/san Bcl6 +/− panels, and 10 µm in the right Roquin san/san Bcl6 +/− panel. (e) Nephritis severity score of 6-mo-old female Roquin +/+ , Roquin san/san , and Roquin san/san Bcl6 +/− mice as determined by histological analysis according to the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Horizontal bars indicate medians. In a, b, and e, each symbol represents one mouse; p-values are indicated on the graphs, and the numbers in the plots represent percentages.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: Heterozygosity for Bcl6 reduces the magnitude of the GC response in Roquin +/+ and Roquin san/san mice and ameliorates the lupus-like phenotype of Roquin san/san mice. (a) Flow cytometric contour plots (left) and graphical analysis (right) of B220 + GL-7 + CD95 + GC B cells in 10-wk-old wild-type ( Bcl6 +/+ ) and Bcl6 +/− mice 8 d after SRBC immunization (P = 0.0011). Data are representative of four independent experiments ( n = 4 per group). (b) Flow cytometric contour plots (left) and dot plots (right) showing B220 + GL-7 + CD95 + GC B cells from 10-wk-old naive Roquin san/san Bcl6 +/+ and Roquin san/san Bcl6 +/− mice. Data are representative of five independent experiments ( n ≥ 4 per group). (c) Representative determination of serum IgG anti-dsDNA from 6-mo-old female Roquin +/+ Bcl6 +/+ , Roquin san/san Bcl6 +/+ , and Roquin san/san Bcl6 +/− mice, determined by immunofluorescence staining of C. luciliae substrate. Data shown reflect the occurrence ( n ≥ 6 mice per group); three out of six Roquin san/san Bcl6 +/− mice had low intensity staining (illustrated in the fourth panel from left), and three out of six were negative (illustrated in the third panel from left). (d) Representative images of kidney sections stained with H E (left) or viewed under an electron microscope (right) from 6-mo-old mice of the indicated genotypes. Roquin san/san animals show widespread mesangial proliferative lesions with moderate interstitial infiltrate. There are multiple electron-dense deposits (arrows). Histological changes in Roquin san/san Bcl6 +/− mice were mild, with occasional electron-dense deposits visible on electron microscopy in two individuals (far right). Images are representative ( n ≥ 4 per group). Bars: (H E) 100 µm in all panels; (electron microscopy) 5 µm in the Roquin +/+ Bcl6 +/+ and Roquin san/san Bcl6 +/+ panels, 2 µm in the left Roquin san/san Bcl6 +/− panels, and 10 µm in the right Roquin san/san Bcl6 +/− panel. (e) Nephritis severity score of 6-mo-old female Roquin +/+ , Roquin san/san , and Roquin san/san Bcl6 +/− mice as determined by histological analysis according to the criteria given in Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081886/DC1 ). Horizontal bars indicate medians. In a, b, and e, each symbol represents one mouse; p-values are indicated on the graphs, and the numbers in the plots represent percentages.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Flow Cytometry, Immunofluorescence, Staining, Microscopy, Electron Microscopy

    T FH cell–associated molecules are decreased in Roquin san/san Sap −/− mice. (a) Representative flow cytometric histograms and (b and c) graphical analysis showing ICOS mean fluorescence intensity (MFI) of splenic naive CD44 low (b) and activated/memory CD44 high (c) CD4 + T cells from 10-wk-old unimmunized mice of the indicated genotypes. Data are representative of three independent experiments. Each symbol represents one mouse. (d) ELISA quantification of IL-21 in supernatant from an overnight culture of splenocytes in the presence of PMA and ionomycin from mice of the indicated genotypes. Data are representative of three experiments. (e) Flow cytometric contour plots of CD40L expression on splenocytes, 5 h after stimulation with anti-CD3 and anti-CD28, derived from mice of the indicated genotypes. (left) Staining with an isotype control; (right) CD40L staining. (f) Histograms of the percentage of CD4 + cells that express CD40L (gated as shown in e) 5 h after CD3 and CD28 stimulation on splenocytes from mice with the indicated genotypes. In d and f, error bars indicate means ± SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Follicular helper T cells are required for systemic autoimmunity

    doi: 10.1084/jem.20081886

    Figure Lengend Snippet: T FH cell–associated molecules are decreased in Roquin san/san Sap −/− mice. (a) Representative flow cytometric histograms and (b and c) graphical analysis showing ICOS mean fluorescence intensity (MFI) of splenic naive CD44 low (b) and activated/memory CD44 high (c) CD4 + T cells from 10-wk-old unimmunized mice of the indicated genotypes. Data are representative of three independent experiments. Each symbol represents one mouse. (d) ELISA quantification of IL-21 in supernatant from an overnight culture of splenocytes in the presence of PMA and ionomycin from mice of the indicated genotypes. Data are representative of three experiments. (e) Flow cytometric contour plots of CD40L expression on splenocytes, 5 h after stimulation with anti-CD3 and anti-CD28, derived from mice of the indicated genotypes. (left) Staining with an isotype control; (right) CD40L staining. (f) Histograms of the percentage of CD4 + cells that express CD40L (gated as shown in e) 5 h after CD3 and CD28 stimulation on splenocytes from mice with the indicated genotypes. In d and f, error bars indicate means ± SEM.

    Article Snippet: A FACSCalibur (BD) with CellQuest software (BD) was used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis.

    Techniques: Mouse Assay, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Staining

    OA-induced activation of PDHs through the regulation of · O 2 − levels by SIRT3. ( a ) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The levels of cellular superoxide anion ( · O 2 − ) and hydrogen peroxide (H 2 O 2 ) were detected by FACSCalibur flow cytometry using the fluorescent dye dihydroethidium at Ex/Em of 300/610 nm or dichlorofluorescein-diacetate at Ex/Em of 488/525 nm, respectively. ( b ) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μ M OA for 10 h under hypoxia, and · O 2 − was detected. ( c , d ) Cells were treated with 100 μ M OA or 10 mM N -acetyl cysteine (antioxidant used as a positive control) in the presence of 1 μ M rotenone for 10 h under hypoxia. The levels of · O 2 − ( c ), protein expression of HIF-OH and HIF1 α ( d ) were detected. ( e , f ) Cells were treated with 100 μ M OA with/without 1 μ M rotenone for 10 h under hypoxia. Cell survival rate ( e ), glucose uptake and production of lactic acid ( f ) were detected, respectively. Bars, S.D.; * P

    Journal: Cell Death & Disease

    Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization

    doi: 10.1038/cddis.2015.86

    Figure Lengend Snippet: OA-induced activation of PDHs through the regulation of · O 2 − levels by SIRT3. ( a ) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The levels of cellular superoxide anion ( · O 2 − ) and hydrogen peroxide (H 2 O 2 ) were detected by FACSCalibur flow cytometry using the fluorescent dye dihydroethidium at Ex/Em of 300/610 nm or dichlorofluorescein-diacetate at Ex/Em of 488/525 nm, respectively. ( b ) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μ M OA for 10 h under hypoxia, and · O 2 − was detected. ( c , d ) Cells were treated with 100 μ M OA or 10 mM N -acetyl cysteine (antioxidant used as a positive control) in the presence of 1 μ M rotenone for 10 h under hypoxia. The levels of · O 2 − ( c ), protein expression of HIF-OH and HIF1 α ( d ) were detected. ( e , f ) Cells were treated with 100 μ M OA with/without 1 μ M rotenone for 10 h under hypoxia. Cell survival rate ( e ), glucose uptake and production of lactic acid ( f ) were detected, respectively. Bars, S.D.; * P

    Article Snippet: The fluorescence intensity was quantified by FACSCalibur flow cytometry (Becton Dickinson) at Ex./Em.=488 nm / 525 nm for H2 O2 detection and Ex/Em=300 nm/610 nm for · O2 − detection, respectively.

    Techniques: Activation Assay, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Incubation, Positive Control, Expressing

    Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by propidium iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P

    Journal: Scientific Reports

    Article Title: Inhibitory effects of isocryptotanshinone on gastric cancer

    doi: 10.1038/s41598-018-27638-0

    Figure Lengend Snippet: Effect of isocryptotanshinone on cell cycle in SGC-7901 ( A ) and MKN-45 ( B ) cells. Cells were serum-starved overnight and treated with the indicated concentration of ICTS and serum for 24 hours, and the contents of DNA stained by propidium iodide were detected using FACScan flow cytometry. The percentages of cells in the sub-G1, G1/G0, S, and G2/M phases were determined by the Flow Jo software. Data were expressed as mean ± standard error of triplicates from a representative experiment. * P

    Article Snippet: The stained cells were analyzed by FACScan flow cytometry (Becton-Dickinson Biosciences) and CellQuest software.

    Techniques: Concentration Assay, Staining, Flow Cytometry, Cytometry, Software

    Effect of isocryptotanshinone on apoptosis of SGC-7901 and MKN-45 cells. Cells were exposed to the indicated concentration of ICTS for 24 hours, and the SGC-7901 ( A ) and MKN-45 ( D ) cells stained using propidium iodide and annexin-v were detected using FACScan flow cytometry. The percentages of apoptotic SGC-7901 ( B ) and MKN-45 ( E ) cells were determined by the Flow Jo software and outputted in histogram. Expression of cleaved PARP and cleaved caspase-9 in SGC-7901 ( C ) and MKN-45 ( F ) cells were determined by western blot. Data were expressed as mean ± standard error of triplicates of one representative experiment. β-actin was used as the loading control. ICTS, isocryptotanshinone.

    Journal: Scientific Reports

    Article Title: Inhibitory effects of isocryptotanshinone on gastric cancer

    doi: 10.1038/s41598-018-27638-0

    Figure Lengend Snippet: Effect of isocryptotanshinone on apoptosis of SGC-7901 and MKN-45 cells. Cells were exposed to the indicated concentration of ICTS for 24 hours, and the SGC-7901 ( A ) and MKN-45 ( D ) cells stained using propidium iodide and annexin-v were detected using FACScan flow cytometry. The percentages of apoptotic SGC-7901 ( B ) and MKN-45 ( E ) cells were determined by the Flow Jo software and outputted in histogram. Expression of cleaved PARP and cleaved caspase-9 in SGC-7901 ( C ) and MKN-45 ( F ) cells were determined by western blot. Data were expressed as mean ± standard error of triplicates of one representative experiment. β-actin was used as the loading control. ICTS, isocryptotanshinone.

    Article Snippet: The stained cells were analyzed by FACScan flow cytometry (Becton-Dickinson Biosciences) and CellQuest software.

    Techniques: Concentration Assay, Staining, Flow Cytometry, Cytometry, Software, Expressing, Western Blot

    Protective effects of eckol, dieckol and 8,8′-bieckol on Aβ 25-35 -indued apoptosis in PC12 cells. ( A ) Morphological apoptosis determined by fluorescence microscopy (400×). ( B ) The percentage of apoptotic cells of the total number of cells. ( C , D ) Early and late apoptosis detected by flow cytometry using Annexin V/7-AAD staining. The lower left area means normal cells (annexin V−/7-AAD−), lower right area means early apoptotic cells (annexin V+/7-AAD−), upper right area means late apopotic and dead cells (annexin V+/7-AAD+) and upper left area means dead cells (annexin V−/7-AAD+). ( E ) Analysis of MMP examined by rhodamine 123. ( F ) The intracellular Ca 2+ levels analyzed using fluo-3AM. ### p

    Journal: Marine Drugs

    Article Title: Anti-Neuroinflammatory Property of Phlorotannins from Ecklonia cava on Aβ25-35-Induced Damage in PC12 Cells

    doi: 10.3390/md17010007

    Figure Lengend Snippet: Protective effects of eckol, dieckol and 8,8′-bieckol on Aβ 25-35 -indued apoptosis in PC12 cells. ( A ) Morphological apoptosis determined by fluorescence microscopy (400×). ( B ) The percentage of apoptotic cells of the total number of cells. ( C , D ) Early and late apoptosis detected by flow cytometry using Annexin V/7-AAD staining. The lower left area means normal cells (annexin V−/7-AAD−), lower right area means early apoptotic cells (annexin V+/7-AAD−), upper right area means late apopotic and dead cells (annexin V+/7-AAD+) and upper left area means dead cells (annexin V−/7-AAD+). ( E ) Analysis of MMP examined by rhodamine 123. ( F ) The intracellular Ca 2+ levels analyzed using fluo-3AM. ### p

    Article Snippet: Flow Cytometry Analysis The cell cycle and apoptosis was determined using the Muse™ Cell Analyzer and Annexin V & Dead Cell Kit (EMD Millipore) according to the manufacturer’s instructions.

    Techniques: Fluorescence, Microscopy, Flow Cytometry, Cytometry, Staining