uea 1 biotin  (Vector Laboratories)

Journal: PLoS ONE

Article Title: Commensal Bacteria Regulate Thymic Aire Expression

doi: 10.1371/journal.pone.0105904

Figure Lengend Snippet: (A) Thymocytes obtained from SPF and GF mice were stained with antibodies against CD4 and CD8. Cell numbers and proportions of CD4/8 subsets were almost equal in the two mice. (B) After roughly enriching CD45 − cells by depleting CD45 + cells from whole thymic cells using AutoMACS, they were stained with anti-I-A d -FITC, CD45-APC and UEA1-biotin and then Streptavidin-PE. Circulated areas in GF and SPF mice in left panels were isolated by FACS Aria to be CD45 − I-A + cells, and they were used for real-time PCR analysis. In separate experiments using 4 GF and SPF mice each, circulated areas were further analyzed for the proportions of UEA1 + I-A + cells in CD45 − I-A + cells. The average proportions for gated UEA1 + I-A + cells in CD45 − I-A + cells from independent experiments are shown in the right panel. (C) CD45 − I-A + cells isolated as TECs from SPF and GF mice were analyzed for Aire mRNA expression by real-time PCR. Relative quantification of gene expression is shown as mean of values from triplicate samples, and the lowest Aire expression level in GF was arbitrarily set at 1. The relative gene expression level is shown for individual TECs from 4 heads each of GF and SPF mice. *P<0.05. (D) For FACS analysis of Aire experiments, enriched CD45 − depleted cells were first stained with CD45-APC and UEA1-biotin facilitated with Streptavidin-PE, and anti-I-A/I-E-Pacific Blue (to distinguish the anti-Aire reaction that was conjugated with Alexa Fluor 488). Then, for intracellular Aire staining, the cells were fixed as described in Materials and Methods and stained with Aire-A488 or control antibody. Representative FACS plots of Aire expression of SPF (black line) and GF (red line) mice are shown. Black and red dotted lines indicate isotype controls of SPF and GF mice, respectively. The mean fluorescence intensity (MFI) for Aire from three independent experiments is shown (right). *P<0.05.

Article Snippet: For flow cytometric analysis and TEC sorting, mouse monoclonal antibodies against CD8- FITC (5H10-1), CD4-PE (RM4-5), CD4-APC (RM4-5), I-A/I-E-Pacific Blue (M5/114.152), and RANKL-biotin (IK22-5) were purchased from Biolegend, CD45-APC (SB1), I-A b -FITC (AF6–120.1), and I-A d -FITC (AMS-32.1) were purchased from BD Bioscience, Aire-Alexa Fluor 488 (5H12), rat IgG2c-Alexa Fluor 488 isotype control (RTK4174), and Streptavidin-PE were purchased from eBioscience, and Ulex Europaeus Agglutinin 1-biotin (UEA1) was purchased from Vector Labs.

Techniques: Staining, Isolation, Real-time Polymerase Chain Reaction, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Commensal Bacteria Regulate Thymic Aire Expression

doi: 10.1371/journal.pone.0105904

Figure Lengend Snippet: (A) Gene expression of TECs isolated from 5-wk-old wild type mice was examined by PCR. (B) From littermates, TECs were isolated individually from 7 heads of Nod1 +/+ (WT) mice and 11 heads of Nod1 −/− mice. mRNA expression was estimated as described in . The lowest Aire or Ctsl expression level in Nod1 −/− mice was arbitrarily set at 1. ***P<0.001. (C) For FACS analysis, the Aire expression on TECs (UEA1 + I-A + CD45 − ) in I-A + CD45 − cells of Nod1 +/+ and Nod1 −/− mice was measured as described in legend. Representative FACS plots of Aire expression of Nod1 +/+ WT (black line) and Nod1 −/− (red line) mice are shown. Black and red dotted lines indicate isotype controls of Nod1 +/+ and Nod1 −/− mice, respectively. MFI for Aire from three independent experiments is shown (right). **P<0.01. (D) Expression of Aire-dependent (Spt1, Expi and S100a8) and -independent (Crp) genes in TECs from Nod1 +/+ (n = 5) and Nod1 −/− (n = 5) mice were analyzed by real-time PCR as described in . *P<0.05, **P<0.01.

Article Snippet: For flow cytometric analysis and TEC sorting, mouse monoclonal antibodies against CD8- FITC (5H10-1), CD4-PE (RM4-5), CD4-APC (RM4-5), I-A/I-E-Pacific Blue (M5/114.152), and RANKL-biotin (IK22-5) were purchased from Biolegend, CD45-APC (SB1), I-A b -FITC (AF6–120.1), and I-A d -FITC (AMS-32.1) were purchased from BD Bioscience, Aire-Alexa Fluor 488 (5H12), rat IgG2c-Alexa Fluor 488 isotype control (RTK4174), and Streptavidin-PE were purchased from eBioscience, and Ulex Europaeus Agglutinin 1-biotin (UEA1) was purchased from Vector Labs.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Commensal Bacteria Regulate Thymic Aire Expression

doi: 10.1371/journal.pone.0105904

Figure Lengend Snippet: (A) E17 BALB/c thymic lobes removed from 3∼5 mothers were separated into right and left lobes and cultured on filter membranes for right and left lobes, and then cultured with and without C12-iE-DAP (10 µg/ml). After 3-day culture and washing, TECs were isolated from thymic lobes by the same procedure as described in adult thymus. Gene expression of the isolated TECs was analyzed by real-time PCR to test Aire expression. In all panels, data were estimated from the relative quantification, and the untreated samples were arbitrarily set at 1. Data bars represent mean ± SD of three independent experiments. *P<0.05. (B) For FACS analysis in the experiment similar to the above (A), Aire expression in cell suspensions obtained from FTOC with and without C12-iE-DAP (10 µg/ml) was manipulated and measured in gated UEA1 + I-A + CD45 − cells by the same procedure as described in legend. Aire expression was clearly increased in TECs receiving C12-iE-DAP (10 µg/ml). For UEA1 + I-A + CD45 − cells, a similar intensity of stained plot in the adult TECs was gated (left). (C) Thymocytes from FTOC stimulated with or without C12-iE-DAP were stained with antibodies against CD4 and CD8. Panels show representative results and percentages of DP and 4SP cells in four independent experiments. (D) After removing lymphocytes by 4-day FTOC culture with dGuo, thymic lobes were washed and re-cultured with C12-iE-DAP (3 and 0.1 µg/ml) for 2 more days. Such manipulated thymus was used to isolate TECs by the same process as described in all experiments. Then, Aire expression of TECs was analyzed by real-time PCR. Data were estimated from the relative quantification, and untreated samples were arbitrarily set at 1. Data bar represents mean ± SD of three independent experiments. *P<0.05. (E) FACS analysis was performed in the same experiment using separate thymi. The staining process for Aire expression in gated UEA1 + I-A + CD45 − cells was similar to that described in . Representative FACS plots and MFI for Aire expression from three independent experiments are shown. **P<0.01.

Article Snippet: For flow cytometric analysis and TEC sorting, mouse monoclonal antibodies against CD8- FITC (5H10-1), CD4-PE (RM4-5), CD4-APC (RM4-5), I-A/I-E-Pacific Blue (M5/114.152), and RANKL-biotin (IK22-5) were purchased from Biolegend, CD45-APC (SB1), I-A b -FITC (AF6–120.1), and I-A d -FITC (AMS-32.1) were purchased from BD Bioscience, Aire-Alexa Fluor 488 (5H12), rat IgG2c-Alexa Fluor 488 isotype control (RTK4174), and Streptavidin-PE were purchased from eBioscience, and Ulex Europaeus Agglutinin 1-biotin (UEA1) was purchased from Vector Labs.

Techniques: Cell Culture, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining