sheep polyclonal antibody against human ido  (Hycult Biotech)

Journal: Journal of Transplantation

Article Title: Peripheral Regulatory Cells Immunophenotyping in Kidney Transplant Recipients with Different Clinical Profiles: A Cross-Sectional Study

doi: 10.1155/2012/256960

Figure Lengend Snippet: Percentage of IDO-and Foxp3-expressing peripheral blood cells in KTR. (a) Representative contour plot from a patient. An electronic gate was made for CD25 hi cells. (b) From the gate a , CD4 + /CD25 hi were determined, and an electronic gate was made for double positive cells. From the latter, CD4 + /CD25 hi /Foxp3 + cells were defined in (c) a healthy donor (HD), (d) a chronic graft dysfunction (CGD), and (k) an excellent long-term graft function under immunosuppression (ELTGF) patient. (f) An electronic gate was made for CCR6 + cells. (g) From the gate f , CCR6 + /CD123 + cells were determined, and an electronic gate was made for double positive cells. From the latter, CCR6 + /CD123 + /IDO + cells were defined in (h) a HD, (i) a CGD patient, and (j) an ELTGF patient. (k, l) Control of FITC-conjugated-rabbit antisheep specificity staining. Percentage of (m) CCR6 + /CD123 hi /IDO + , (n) CD4 + /CD25 hi /Foxp3 + , and (o) CD8 + /CD28 − /Foxp3 + peripheral blood cells. A total of 50,000 events were recorded for each sample before any gate setting and analyzed with the CellQuestPro software (BD Biosciences). Results are expressed as median, 10th, 25th, 75th, and 90th percentiles.

Article Snippet: Cells were stained with a sheep anti-human-IDO (Chemicon, Temecula, CA, USA) and then with FITC-conjugated-rabbit antisheep antibody.

Techniques: Expressing, Staining, Software

Journal: PLoS ONE

Article Title: M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

doi: 10.1371/journal.pone.0037314

Figure Lengend Snippet: (A) Microarray results of BAL cells (>90% macrophages) infected with M. tuberculosis (MOI 3–5) for 24 h compared to uninfected cells. Increased mRNA expression levels for Indoleamine 2,3-dioxygenase (IDO-1), Kynurenine 3-monooxygenase (KYN-OX), and L-Kynurenine hydrolase (KYN-HYD) are depicted as fold induction upon M. tuberculosis infection compared to uninfected control cells. Data are means +/− standard error of comparisons of microarray data obtained from four different individuals. (B) Murine bone marrow-derived macrophages were left untreated (1), infected with M. tuberculosis (MOI 5) (2) and stimulated with IFN-γ (100 U/ml) (3). Co-stimulation with both M. tuberculosis and IFN-γ was either performed simultaneously (4) or cells were pre-treated with IFN-γ for 24 h and then infected with M. tuberculosis for 24 h (5) before RNA isolation. IDO-1 mRNA expression and beta-2-microglobulin (control) were detected at 4, 24, and 48 h by RT-PCR. (6) H 2 O. Data are representative of two independent experiments.

Article Snippet: Proteins from the gels were transferred onto nitrocellulose membranes using a Bio-Rad Mini Transblot system for 1 h at 140 V. The membranes were blocked with PBS/5% skim milk powder overnight at 4°C, washed and incubated with sheep anti-human IDO-1 antibody (Chemicon International), followed by washing and incubation with rabbit anti-sheep IgG (Invitrogen), then donkey anti-rabbit IgG conjugated to HRP (Amersham, Life Science) all in PBS/1% BSA for 1 h at RT.

Techniques: Microarray, Infection, Expressing, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

doi: 10.1371/journal.pone.0037314

Figure Lengend Snippet: Bone marrow-derived DCs were cultured for 4 days in vitro before stimulation for 2 h with Ag85 peptide P25. DCs were overlaid with CFSE labelled CD4 + T cells from P25-specific T cell receptor transgenic mice. Proliferation of T cells was determined by flow cytometry. DCs from WT and IDO-1 −/− mice were compared in their ability to induce proliferation of P25-specific T cells after 2 days (A, D) and 5 days (B, E). WT DCs were treated with 1 Methyl-DL-tryptophan before stimulation with P25 peptide. P25-specific T cell proliferation was determined after 5 days (C, F). (A-C) Fluorescence intensities of representative FACS plots are depicted. Grey- IDO-1 −/− /1-Methyl-DL-tryptophan, Black – WT/untreated control. The percentages of cells per division were calculated (D-F). Data represent the mean +/− SD of triplicate cultures from one of three representative experiments. The differences between groups were analysed by analysis of variance, *P<0.001.

Article Snippet: Proteins from the gels were transferred onto nitrocellulose membranes using a Bio-Rad Mini Transblot system for 1 h at 140 V. The membranes were blocked with PBS/5% skim milk powder overnight at 4°C, washed and incubated with sheep anti-human IDO-1 antibody (Chemicon International), followed by washing and incubation with rabbit anti-sheep IgG (Invitrogen), then donkey anti-rabbit IgG conjugated to HRP (Amersham, Life Science) all in PBS/1% BSA for 1 h at RT.

Techniques: Derivative Assay, Cell Culture, In Vitro, Transgenic Assay, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

doi: 10.1371/journal.pone.0037314

Figure Lengend Snippet: WT and IDO-1 −/− mice were infected with M. tuberculosis (100 CFU). Single cell suspensions were prepared from infected lungs and analyzed by flow cytometry for (A) CD4+, (B) CD8+ cells, and the percentages of these cells with an activated CD44hi/CD62lo phenotype ((C) CD4+, (D) CD8+). (E) Numbers of IFN-γ producing cells determined by ELISpot for lung cells cultured at 1×10 5 cells/well with M. tuberculosis CFP overnight. Data represent the mean +/− SD of 5 mice from one representative of two experiments.

Article Snippet: Proteins from the gels were transferred onto nitrocellulose membranes using a Bio-Rad Mini Transblot system for 1 h at 140 V. The membranes were blocked with PBS/5% skim milk powder overnight at 4°C, washed and incubated with sheep anti-human IDO-1 antibody (Chemicon International), followed by washing and incubation with rabbit anti-sheep IgG (Invitrogen), then donkey anti-rabbit IgG conjugated to HRP (Amersham, Life Science) all in PBS/1% BSA for 1 h at RT.

Techniques: Infection, Flow Cytometry, Enzyme-linked Immunospot, Cell Culture

Journal: PLoS ONE

Article Title: M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

doi: 10.1371/journal.pone.0037314

Figure Lengend Snippet: (A) Bone marrow-derived macrophages of WT and IDO-1 −/− mice were infected with M. tuberculosis (MOI 1) in the presence of IFN-γ (100 U/ml). Intracellular bacterial numbers were determined at the indicated time points. Data points are means +/− SD of triplicate wells of one representative of 3 independent experiments. (B) Human BAL cells were stimulated with 100 U/ml IFN-γ and infected with M. tuberculosis (MOI 1) in the presence or absence of 1-Methyl-DL-Tryptophan (250 µM). Bacterial counts in cell lysates were determined at the times indicated. Data points are means +/− SD of triplicate wells of one representative of 6 independent experiments.

Article Snippet: Proteins from the gels were transferred onto nitrocellulose membranes using a Bio-Rad Mini Transblot system for 1 h at 140 V. The membranes were blocked with PBS/5% skim milk powder overnight at 4°C, washed and incubated with sheep anti-human IDO-1 antibody (Chemicon International), followed by washing and incubation with rabbit anti-sheep IgG (Invitrogen), then donkey anti-rabbit IgG conjugated to HRP (Amersham, Life Science) all in PBS/1% BSA for 1 h at RT.

Techniques: Derivative Assay, Infection

Journal: PLoS ONE

Article Title: M. tuberculosis Induces Potent Activation of IDO-1, but This Is Not Essential for the Immunological Control of Infection

doi: 10.1371/journal.pone.0037314

Figure Lengend Snippet: WT and IDO-1 −/− mice were infected with M. tuberculosis (100 CFU). IDO-1 expression in the lungs of uninfected WT mice (A), and WT (B) and IDO-1 −/− (C) mice at 28 days post infection. Lungs from uninfected IDO-1 −/− mice showed no IDO-1-positive cells (data not shown). IDO-1-expression in lungs of WT mice was primarily associated with inflammatory lesions within large monocytic cells (B), while IDO-1 −/− mice show no specific staining (C). One representative section from 1 of 5 mice per group. Bacterial loads (D) in infected lungs were determined at the time points indicated. Data represent the means +/− SD of 5 mice per group from one of two independent experiments. Lung inflammation (E) as percentage of the lung area with inflammatory cell infiltration from WT and IDO-1 −/− mice after subtracting the background cellularity in normal lung. To assess survival (F), infected mice were monitored daily and euthanized when showing signs of ill health. Data represent the time to euthanasia of 6 mice per group.

Article Snippet: Proteins from the gels were transferred onto nitrocellulose membranes using a Bio-Rad Mini Transblot system for 1 h at 140 V. The membranes were blocked with PBS/5% skim milk powder overnight at 4°C, washed and incubated with sheep anti-human IDO-1 antibody (Chemicon International), followed by washing and incubation with rabbit anti-sheep IgG (Invitrogen), then donkey anti-rabbit IgG conjugated to HRP (Amersham, Life Science) all in PBS/1% BSA for 1 h at RT.

Techniques: Infection, Expressing, Staining