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  • 92
    BEI Resources m tuberculosis
    M Tuberculosis, supplied by BEI Resources, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco m tuberculosis
    M Tuberculosis, supplied by Difco, used in various techniques. Bioz Stars score: 96/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson m tuberculosis
    M Tuberculosis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 97/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore m tuberculosis
    M Tuberculosis, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chondrex inc m tuberculosis
    M Tuberculosis, supplied by Chondrex inc, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corixa m tuberculosis
    M Tuberculosis, supplied by Corixa, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Connaught Labs m tuberculosis
    M Tuberculosis, supplied by Connaught Labs, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript m tuberculosis
    M Tuberculosis, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore m tuberculosis tac
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    Difco m tuberculosis h37ra
    Lipid antigen specificity of J.RT3/α/β TCR transfectants. CD8-2/J.RT3 (top), DN1/J.RT3 (middle), or CD8-1/J.RT3 (bottom) transfectants (10 5 /well) were cultured in triplicate for 24 h in the presence of PMA (10 ng/ml) and CD1 + monocytes (5 × 10 4 /well) with no antigen (asterisk), or with a titration of M. tuberculosis <t>H37Ra</t> mycolic acids (▪), silica fraction 60:40 (▵), or silica fraction 90:10 (•). To measure IL-2 production by the transfectants, an aliquot of the supernatants was removed and diluted 1:4 with medium. HT-2 cells (5 × 10 3 /well) were added to the supernatants and cultured for 16–24 h. [ 3 H]Thymidine was added during the final 5–6 h culture, after which the plates were harvested and [ 3 H]thymidine incorporation measured with a liquid scintillation counter. Results are expressed as the mean cpm ± SD of triplicate cultures.
    M Tuberculosis H37ra, supplied by Difco, used in various techniques. Bioz Stars score: 91/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis erdman
    Protection conferred to infection with M. tuberculosis by BCG SSI and BCG Pasteur Aeras. Groups of 6 <t>C57BL/6</t> mice were infected 6 weeks after immunization with BCG SSI (grey squares) or BCG Pateur Aeras (grey circles) via the intranasal route with 700 CFU M. tuberculosis <t>Erdman.</t> Control animals received saline at the time of immunization (open squares). CFUs per organ were determined 4 weeks after infection in lungs ( a ) and spleens ( b ). Each data point represents an individual animal. Error bars represent the median +/- interquartile range. Statistical significance was determined by one-way ANOVA with Holm-Sidak correction for multiple comparisons using GraphPad Prism
    M Tuberculosis Erdman, supplied by BEI Resources, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson m tuberculosis h37ra
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis H37ra, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis cdc1551
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis Cdc1551, supplied by BEI Resources, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gen-Probe m tuberculosis complex
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis Complex, supplied by Gen-Probe, used in various techniques. Bioz Stars score: 88/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources irradiated m tuberculosis
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    Irradiated M Tuberculosis, supplied by BEI Resources, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis lysate
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis Lysate, supplied by BEI Resources, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher m tuberculosis rho
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis Rho, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis cfp
    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the <t>H37Ra</t> strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p
    M Tuberculosis Cfp, supplied by BEI Resources, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pasteur Institute m tuberculosis h37rv
    Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and <t>H37Rv</t> evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .
    M Tuberculosis H37rv, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 91/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis h37rv
    DCP activity against M. tuberculosis <t>H37Rv.</t> PBMCs were plated at a density of 1.5 × 10 6 cells per well in round-bottom 96-well plates and incubated overnight at 37°C. After 2 h of incubation, nonadherent cells were washed off, and the
    M Tuberculosis H37rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources m tuberculosis hn878
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
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    Gen-Probe accuprobe m tuberculosis test
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    Accuprobe M Tuberculosis Test, supplied by Gen-Probe, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sigma-Genosys panaroma m tuberculosis microarray
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    Panaroma M Tuberculosis Microarray, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gen-Probe m tuberculosis direct test
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
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    Roche amplicor m tuberculosis test
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    Amplicor M Tuberculosis Test, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources irradiated m tuberculosis h37rv
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
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    Hologic Inc m tuberculosis direct test
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
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    Abbott Laboratories lcx m tuberculosis assay
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    Lcx M Tuberculosis Assay, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 84/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Glas-Col virulent m tuberculosis h37rv
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    Virulent M Tuberculosis H37rv, supplied by Glas-Col, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc m tuberculosis
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    M Tuberculosis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals m tuberculosis
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
    M Tuberculosis, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gen-Probe m tuberculosis
    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis <t>HN878.</t> Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed
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    Lipid antigen specificity of J.RT3/α/β TCR transfectants. CD8-2/J.RT3 (top), DN1/J.RT3 (middle), or CD8-1/J.RT3 (bottom) transfectants (10 5 /well) were cultured in triplicate for 24 h in the presence of PMA (10 ng/ml) and CD1 + monocytes (5 × 10 4 /well) with no antigen (asterisk), or with a titration of M. tuberculosis H37Ra mycolic acids (▪), silica fraction 60:40 (▵), or silica fraction 90:10 (•). To measure IL-2 production by the transfectants, an aliquot of the supernatants was removed and diluted 1:4 with medium. HT-2 cells (5 × 10 3 /well) were added to the supernatants and cultured for 16–24 h. [ 3 H]Thymidine was added during the final 5–6 h culture, after which the plates were harvested and [ 3 H]thymidine incorporation measured with a liquid scintillation counter. Results are expressed as the mean cpm ± SD of triplicate cultures.

    Journal: The Journal of Experimental Medicine

    Article Title: Molecular Recognition of Lipid Antigens by T Cell Receptors

    doi:

    Figure Lengend Snippet: Lipid antigen specificity of J.RT3/α/β TCR transfectants. CD8-2/J.RT3 (top), DN1/J.RT3 (middle), or CD8-1/J.RT3 (bottom) transfectants (10 5 /well) were cultured in triplicate for 24 h in the presence of PMA (10 ng/ml) and CD1 + monocytes (5 × 10 4 /well) with no antigen (asterisk), or with a titration of M. tuberculosis H37Ra mycolic acids (▪), silica fraction 60:40 (▵), or silica fraction 90:10 (•). To measure IL-2 production by the transfectants, an aliquot of the supernatants was removed and diluted 1:4 with medium. HT-2 cells (5 × 10 3 /well) were added to the supernatants and cultured for 16–24 h. [ 3 H]Thymidine was added during the final 5–6 h culture, after which the plates were harvested and [ 3 H]thymidine incorporation measured with a liquid scintillation counter. Results are expressed as the mean cpm ± SD of triplicate cultures.

    Article Snippet: CD8-2/J.RT3 cells produced IL-2 in a dose-dependent manner when cultured with the appropriate lipid fraction of M. tuberculosis H37Ra (silica fraction 90:10; see Materials and Methods), but not with other lipid preparations that did not contain the specific antigen recognized by CD8-2 (silica fraction 60:40 or purified mycolic acids) (Fig. top).

    Techniques: Cell Culture, Titration

    CD1 specificity of J.RT3/α/β TCR transfectants. CD8-2/ J.RT3 (A), DN1/J.RT3 (B), or CD8-1/J.RT3 (C) transfectants (10 5 / well) were cultured in triplicate for 24 h in the presence of PMA (10 ng/ml) and CD1 + monocytes (5 × 10 4 /well) with no antigen, or with 50 μg/ml M. tuberculosis H37Ra mycolic acids (DN1/J.RT3), 1 μg/ml silica fraction 60:40 (CD8-1/J.RT3), or 1 μg/ml silica fraction 90:10 (CD8-2/ J.RT3). Cultures with antigen were set up with or without the following mAbs at a final concentration of 20 μg/ml: 10H3.9.3 (anti-CD1a), BCD1b3.1 (anti-CD1b), or F10/21A3.8 (anti-CD1c). IL-2 production was measured as described for Fig. 3 . Results are expressed as the mean cpm ± SD of triplicate cultures.

    Journal: The Journal of Experimental Medicine

    Article Title: Molecular Recognition of Lipid Antigens by T Cell Receptors

    doi:

    Figure Lengend Snippet: CD1 specificity of J.RT3/α/β TCR transfectants. CD8-2/ J.RT3 (A), DN1/J.RT3 (B), or CD8-1/J.RT3 (C) transfectants (10 5 / well) were cultured in triplicate for 24 h in the presence of PMA (10 ng/ml) and CD1 + monocytes (5 × 10 4 /well) with no antigen, or with 50 μg/ml M. tuberculosis H37Ra mycolic acids (DN1/J.RT3), 1 μg/ml silica fraction 60:40 (CD8-1/J.RT3), or 1 μg/ml silica fraction 90:10 (CD8-2/ J.RT3). Cultures with antigen were set up with or without the following mAbs at a final concentration of 20 μg/ml: 10H3.9.3 (anti-CD1a), BCD1b3.1 (anti-CD1b), or F10/21A3.8 (anti-CD1c). IL-2 production was measured as described for Fig. 3 . Results are expressed as the mean cpm ± SD of triplicate cultures.

    Article Snippet: CD8-2/J.RT3 cells produced IL-2 in a dose-dependent manner when cultured with the appropriate lipid fraction of M. tuberculosis H37Ra (silica fraction 90:10; see Materials and Methods), but not with other lipid preparations that did not contain the specific antigen recognized by CD8-2 (silica fraction 60:40 or purified mycolic acids) (Fig. top).

    Techniques: Cell Culture, Concentration Assay

    Protection conferred to infection with M. tuberculosis by BCG SSI and BCG Pasteur Aeras. Groups of 6 C57BL/6 mice were infected 6 weeks after immunization with BCG SSI (grey squares) or BCG Pateur Aeras (grey circles) via the intranasal route with 700 CFU M. tuberculosis Erdman. Control animals received saline at the time of immunization (open squares). CFUs per organ were determined 4 weeks after infection in lungs ( a ) and spleens ( b ). Each data point represents an individual animal. Error bars represent the median +/- interquartile range. Statistical significance was determined by one-way ANOVA with Holm-Sidak correction for multiple comparisons using GraphPad Prism

    Journal: BMC Infectious Diseases

    Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

    doi: 10.1186/s12879-016-1751-4

    Figure Lengend Snippet: Protection conferred to infection with M. tuberculosis by BCG SSI and BCG Pasteur Aeras. Groups of 6 C57BL/6 mice were infected 6 weeks after immunization with BCG SSI (grey squares) or BCG Pateur Aeras (grey circles) via the intranasal route with 700 CFU M. tuberculosis Erdman. Control animals received saline at the time of immunization (open squares). CFUs per organ were determined 4 weeks after infection in lungs ( a ) and spleens ( b ). Each data point represents an individual animal. Error bars represent the median +/- interquartile range. Statistical significance was determined by one-way ANOVA with Holm-Sidak correction for multiple comparisons using GraphPad Prism

    Article Snippet: Infection of mice with M. tuberculosis Female C57BL/6 mice were infected intranasally with M. tuberculosis Erdman (BEI Resources, Manassas, VA, USA) 6 weeks after immunization and kept in isolators under BSL-3 containment.

    Techniques: Infection, Mouse Assay

    Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the H37Ra strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p

    Journal: Emerging Microbes & Infections

    Article Title: Acetylation of lysine 182 inhibits the ability of Mycobacterium tuberculosis DosR to bind DNA and regulate gene expression during hypoxia

    doi: 10.1038/s41426-018-0112-3

    Figure Lengend Snippet: Acetylation of DosR K182 affects gene expression of the DosR regulon in vivo. a Acetylation pattern of K182 in DosR from WT and KO mutant under aerobic or hypoxic conditions. The WT and KO mutant bacteria were cultured in Dubos medium at 37 °C for 2 days. The aerobic cultures were grown in 50-ml tubes (10 ml of culture), and hypoxic cultures were grown in 50-ml wax-sealed bottles with a headspace-to-medium ratio of 0.5. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-b, in Supplementary material). Results are representative of three independent experiments. b The transcript levels of candidate genes in the H37Ra strain and KO mutant under aerobic or hypoxic conditions. RNA was isolated from the strains grown in Dubos medium under aerobic or hypoxic conditions (see above), and the transcript levels of the candidate genes were determined by qPCR using the method of 2 −ΔΔCt . The data were normalized to rrs and compared to WT. Two-way ANOVA was performed to compare WT and KO in each condition; * p

    Article Snippet: M. tuberculosis H37Ra and the MRA_1161 deletion-mutant strain (∆ npdA ) were inoculated into 7H9–OADC–0.05% Tween 80 medium (Becton Dickinson, Sparks, MD, USA) and cultured at 37 °C for 7 days until the log phase (OD600 0.4–0.6) was reached.

    Techniques: Expressing, In Vivo, Mutagenesis, Cell Culture, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Isolation, Real-time Polymerase Chain Reaction

    DosR can be deacetylated in vitro and in vivo. a Rv1151c deacetylates DosR in a dose-dependent manner in vitro. Purified DosR 182Ac (8 µM) was incubated with NAD + (1 mM) and Rv1151c at the indicated concentrations (1.5 µM, 3 µM, and 6 µM) at 25 °C for 2 h. The acetylation levels were evaluated by western blot with anti-DosR (1:10,000) at 3-s exposure and anti-K182 Ac (1:1000) at 30-s exposure. The results are representative of three independent experiments. b Deacetylation of DosR is dependent on NAD + and is inhibited by NAM. Purified DosR 182Ac (8 µM) was incubated with or without Rv1151c (6 µM), NAD + (1 mM), and NAM (10 mM) at 25 °C for 2 h. The acetylation levels were evaluated by western blotting with anti-DosR (1:10,000) at 3-s exposure and anti-K182 Ac (1:1000) at 30-s exposure. Results are representative of three independent experiments. c The acetylation levels of DosR in H37Ra wild-type (Ra) and MRA_1161 ( Rv1151c , homolog in H37Ra)-deletion mutant (KO) in vivo. H37Ra and KO were inoculated into 7H9–10% OADC–0.05% Tween 80 medium and grown to mid-log phase (OD 600 = 0.4–0.6), respectively. The cultures were incubated with or without NAM (5 mM) for 10 h and collected by centrifugation. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-a, in Supplementary material). Results are representative of three independent experiments

    Journal: Emerging Microbes & Infections

    Article Title: Acetylation of lysine 182 inhibits the ability of Mycobacterium tuberculosis DosR to bind DNA and regulate gene expression during hypoxia

    doi: 10.1038/s41426-018-0112-3

    Figure Lengend Snippet: DosR can be deacetylated in vitro and in vivo. a Rv1151c deacetylates DosR in a dose-dependent manner in vitro. Purified DosR 182Ac (8 µM) was incubated with NAD + (1 mM) and Rv1151c at the indicated concentrations (1.5 µM, 3 µM, and 6 µM) at 25 °C for 2 h. The acetylation levels were evaluated by western blot with anti-DosR (1:10,000) at 3-s exposure and anti-K182 Ac (1:1000) at 30-s exposure. The results are representative of three independent experiments. b Deacetylation of DosR is dependent on NAD + and is inhibited by NAM. Purified DosR 182Ac (8 µM) was incubated with or without Rv1151c (6 µM), NAD + (1 mM), and NAM (10 mM) at 25 °C for 2 h. The acetylation levels were evaluated by western blotting with anti-DosR (1:10,000) at 3-s exposure and anti-K182 Ac (1:1000) at 30-s exposure. Results are representative of three independent experiments. c The acetylation levels of DosR in H37Ra wild-type (Ra) and MRA_1161 ( Rv1151c , homolog in H37Ra)-deletion mutant (KO) in vivo. H37Ra and KO were inoculated into 7H9–10% OADC–0.05% Tween 80 medium and grown to mid-log phase (OD 600 = 0.4–0.6), respectively. The cultures were incubated with or without NAM (5 mM) for 10 h and collected by centrifugation. Cell extracts (20 μg per lane) were analyzed by western blotting with anti-DosR (1:10,000) at 30-s exposure and anti-K182 Ac (1:1000) at 10-min exposure. Equal amounts of loading control for western blotting were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue (Fig. S-a, in Supplementary material). Results are representative of three independent experiments

    Article Snippet: M. tuberculosis H37Ra and the MRA_1161 deletion-mutant strain (∆ npdA ) were inoculated into 7H9–OADC–0.05% Tween 80 medium (Becton Dickinson, Sparks, MD, USA) and cultured at 37 °C for 7 days until the log phase (OD600 0.4–0.6) was reached.

    Techniques: In Vitro, In Vivo, Purification, Incubation, Western Blot, Mutagenesis, Centrifugation, Polyacrylamide Gel Electrophoresis, Staining

    Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and H37Rv evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and H37Rv evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Quantitative RT-PCR, Expressing

    Secretion Analysis In vitro expression and secretion of ESAT-6 from M. tuberculosis H37Rv, H37Ra, MT103 phoP ko (SO2), and recombinant H37Ra complemented with integrating cosmids carrying genes phoP or rpsL from H37Rv. Total protein concentrations were determined by using Bio-Rad protein assay, and 15-μg samples were subjected to SDS-PAGE. Detection was carried out by using monoclonal anti-ESAT-6 antibody Hyb 76–8a.

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Secretion Analysis In vitro expression and secretion of ESAT-6 from M. tuberculosis H37Rv, H37Ra, MT103 phoP ko (SO2), and recombinant H37Ra complemented with integrating cosmids carrying genes phoP or rpsL from H37Rv. Total protein concentrations were determined by using Bio-Rad protein assay, and 15-μg samples were subjected to SDS-PAGE. Detection was carried out by using monoclonal anti-ESAT-6 antibody Hyb 76–8a.

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: In Vitro, Expressing, Recombinant, SDS Page

    Antigen-Specific IL-2 Production of T Cell Hybridomas Analysis of IL-2 secretion by anti-ESAT-6 or anti-Ag85A T cell hybridomas in response to recognition of antigen presented by bone marrow–derived dendritic cells (BM-DC) incubated with homologous or control peptides or infected with H37Rv, H37Ra or recombinant H37Ra strains.

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Antigen-Specific IL-2 Production of T Cell Hybridomas Analysis of IL-2 secretion by anti-ESAT-6 or anti-Ag85A T cell hybridomas in response to recognition of antigen presented by bone marrow–derived dendritic cells (BM-DC) incubated with homologous or control peptides or infected with H37Rv, H37Ra or recombinant H37Ra strains.

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Derivative Assay, Incubation, Infection, Recombinant

    Macrophage Infection Studies Bone marrow–derived murine macrophages (BMM) were infected with M. tuberculosis H37Ra, H37Ra:: fadE5 , H37Ra:: phoP , and H37Rv at a MOI of ca. 1:1 and 10:1 bacteria per cell (A and B, respectively). The figure shows the means and standard deviations of CFU ratio values (CFU at days 1, 4, and 7 relative to CFU at 4 h) obtained in a representative experiment performed in quadruplicate. The significant difference levels in growth characteristics between H37Ra and other strains (H37Ra:: phoP and H37Rv) were determined by analysis of variance (ANOVA) (* p

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Macrophage Infection Studies Bone marrow–derived murine macrophages (BMM) were infected with M. tuberculosis H37Ra, H37Ra:: fadE5 , H37Ra:: phoP , and H37Rv at a MOI of ca. 1:1 and 10:1 bacteria per cell (A and B, respectively). The figure shows the means and standard deviations of CFU ratio values (CFU at days 1, 4, and 7 relative to CFU at 4 h) obtained in a representative experiment performed in quadruplicate. The significant difference levels in growth characteristics between H37Ra and other strains (H37Ra:: phoP and H37Rv) were determined by analysis of variance (ANOVA) (* p

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Infection, Derivative Assay

    DCP activity against M. tuberculosis H37Rv. PBMCs were plated at a density of 1.5 × 10 6 cells per well in round-bottom 96-well plates and incubated overnight at 37°C. After 2 h of incubation, nonadherent cells were washed off, and the

    Journal: Infection and Immunity

    Article Title: Dipterinyl Calcium Pentahydrate Inhibits Intracellular Mycobacterial Growth in Human Monocytes via the C-C Chemokine MIP-1? and Nitric Oxide

    doi: 10.1128/IAI.01393-12

    Figure Lengend Snippet: DCP activity against M. tuberculosis H37Rv. PBMCs were plated at a density of 1.5 × 10 6 cells per well in round-bottom 96-well plates and incubated overnight at 37°C. After 2 h of incubation, nonadherent cells were washed off, and the

    Article Snippet: M. tuberculosis H37Rv (catalog number NR-123) was obtained from BEI Resources, Manassas, VA. Before being used for infection, bacteria were thawed and sonicated for l min in a cup sonicator (W385; Heat Systems Ultrasonics, Farmingdale, NY) to obtain a single-cell suspension and diluted appropriately in antibiotic-free complete RPMI 1640 medium.

    Techniques: Activity Assay, Incubation

    Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis HN878. Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed

    Journal: Journal of Bacteriology

    Article Title: Interaction of Erp Protein of Mycobacterium tuberculosis with Rv2212 Enhances Intracellular Survival of Mycobacterium smegmatis

    doi: 10.1128/JB.00120-16

    Figure Lengend Snippet: Immunodetection and interaction of RvErp and Rv2212 at the endogenous level. (A) Immunodetection of Rv2212 and RvErp in subcellular fractions of M. tuberculosis HN878. Cell fractions of M. tuberculosis HN878 obtained from BEI Resources were electrophoresed

    Article Snippet: The subcellular fractions of M. tuberculosis HN878 were obtained from BEI Resources and electrophoresed on SDS-PAGE followed by transfer onto nitrocellulose membrane.

    Techniques: Immunodetection