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  • 99
    Thermo Fisher remel oadc enrichment
    Remel Oadc Enrichment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/remel oadc enrichment/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    remel oadc enrichment - by Bioz Stars, 2020-04
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    99
    Millipore oadc supplement
    Oadc Supplement, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 19 article reviews
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    oadc supplement - by Bioz Stars, 2020-04
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    98
    Becton Dickinson oadc
    Growth kinetics of M. tuberculosis (H37Rv), echA5 mutant and fadB3 mutant in (a) <t>Middlebrook</t> <t>7H9-Tw-Glycerol-OADC</t> medium and (b) Sauton’s minimal medium plus 0.2% glycerol. Results shown are the average of three independent experiments. Error bars represent SD.
    Oadc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 98/100, based on 904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 904 article reviews
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    oadc - by Bioz Stars, 2020-04
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    oadc  (Difco)
    94
    Difco oadc
    Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo . Exponentially growing M. tuberculosis mc 2 <t>6230</t> was incubated with increasing concentrations of CyC 17 or CyC 7β in 7H9 <t>OADC/Tween</t> 80 at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2- 14 C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A , chemical structures of the CyC analogs used in this study. B and C , effect of CyC 17 ( B ) or CyC 7β ( C ) treatment on the mycolic acid profiles of M. tuberculosis mc 2 6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( far left ). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) ( middle left ). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( middle right ). Densitometric analysis ( far right ) was performed on the TLCs shown in the left panels. Histograms and error bars , means and S.D. values calculated from at least two independent experiments.
    Oadc, supplied by Difco, used in various techniques. Bioz Stars score: 94/100, based on 334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 334 article reviews
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    oadc - by Bioz Stars, 2020-04
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    93
    MiddleBrook Pharmaceuticals oadc
    Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo . Exponentially growing M. tuberculosis mc 2 <t>6230</t> was incubated with increasing concentrations of CyC 17 or CyC 7β in 7H9 <t>OADC/Tween</t> 80 at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2- 14 C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A , chemical structures of the CyC analogs used in this study. B and C , effect of CyC 17 ( B ) or CyC 7β ( C ) treatment on the mycolic acid profiles of M. tuberculosis mc 2 6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( far left ). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) ( middle left ). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( middle right ). Densitometric analysis ( far right ) was performed on the TLCs shown in the left panels. Histograms and error bars , means and S.D. values calculated from at least two independent experiments.
    Oadc, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 93/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    oadc - by Bioz Stars, 2020-04
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    94
    Thermo Fisher oadc
    Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo . Exponentially growing M. tuberculosis mc 2 <t>6230</t> was incubated with increasing concentrations of CyC 17 or CyC 7β in 7H9 <t>OADC/Tween</t> 80 at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2- 14 C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A , chemical structures of the CyC analogs used in this study. B and C , effect of CyC 17 ( B ) or CyC 7β ( C ) treatment on the mycolic acid profiles of M. tuberculosis mc 2 6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( far left ). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) ( middle left ). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( middle right ). Densitometric analysis ( far right ) was performed on the TLCs shown in the left panels. Histograms and error bars , means and S.D. values calculated from at least two independent experiments.
    Oadc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 89 article reviews
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    97
    Millipore oadc
    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in <t>7H9</t> culture medium supplemented with <t>OADC</t> and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).
    Oadc, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Hardy Diagnostics oadc
    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in <t>7H9</t> culture medium supplemented with <t>OADC</t> and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).
    Oadc, supplied by Hardy Diagnostics, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    HiMedia Laboratories oadc
    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in <t>7H9</t> culture medium supplemented with <t>OADC</t> and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).
    Oadc, supplied by HiMedia Laboratories, 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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    91
    US Biological Life Sciences oadc
    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in <t>7H9</t> culture medium supplemented with <t>OADC</t> and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).
    Oadc, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson 7h11 oadc
    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in <t>7H9</t> culture medium supplemented with <t>OADC</t> and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).
    7h11 Oadc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    7h11 oadc - by Bioz Stars, 2020-04
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    93
    Difco oadc enrichment
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Enrichment, supplied by Difco, used in various techniques. Bioz Stars score: 93/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Difco middlebrook oadc
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Middlebrook Oadc, supplied by Difco, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Becton Dickinson oadc supplement
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Supplement, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 96/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    HiMedia Laboratories oadc supplement
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Supplement, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson middlebook oadc
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Middlebook Oadc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    MiddleBrook Pharmaceuticals oadc enrichment
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Enrichment, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Yorlab oadc supplement
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Supplement, supplied by Yorlab, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific oadc enrichment
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Enrichment, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Difco oadc supplement
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Supplement, supplied by Difco, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Merck & Co oadc supplement
    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto <t>Middlebrook</t> 7H10 plates containing <t>OADC,</t> glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.
    Oadc Supplement, supplied by Merck & Co, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Difco 7h10 oadc
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    Becton Dickinson mls oadc
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    Thermo Fisher oadc broth
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    Becton Dickinson catalase oadc
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    Hardy Diagnostics catalase oadc
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
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    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in <t>7H9</t> <t>OADC</t> containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.
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    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in <t>7H9</t> <t>OADC</t> containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.
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    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in <t>7H9</t> <t>OADC</t> containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.
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    Image Search Results


    Growth kinetics of M. tuberculosis (H37Rv), echA5 mutant and fadB3 mutant in (a) Middlebrook 7H9-Tw-Glycerol-OADC medium and (b) Sauton’s minimal medium plus 0.2% glycerol. Results shown are the average of three independent experiments. Error bars represent SD.

    Journal: Tuberculosis (Edinburgh, Scotland)

    Article Title: The Mycobacterium tuberculosis ?-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo

    doi: 10.1016/j.tube.2011.06.006

    Figure Lengend Snippet: Growth kinetics of M. tuberculosis (H37Rv), echA5 mutant and fadB3 mutant in (a) Middlebrook 7H9-Tw-Glycerol-OADC medium and (b) Sauton’s minimal medium plus 0.2% glycerol. Results shown are the average of three independent experiments. Error bars represent SD.

    Article Snippet: 2.1 Bacterial strains and growth conditions M. tuberculosis H37 Rv was grown in Middlebrook 7H9-Tw-Glycerol-OADC liquid broth (supplemented with 0.2% glycerol, 0.05% Tween-80 and 10% OADC) at 37 °C shaken at 150 r.p.m. in an orbital shaker, on Middlebrook 7H11 solid media supplemented with 0.5% glycerol and 10% OADC (Becton Dickinson) or in Sauton’s Minimal Medium supplemented with 0.2% glycerol, 0.0001% ZnSO4 and 0.025% Tyloxapol as described previously.

    Techniques: Mutagenesis

    Deletion of MSMEG_1285 in Msmeg mc 2 155 impacts the cell-surface exposure of HBHA_ Mtb . AFM tomographic image (left panel), representative spatially-resolved map of adhesion forces recorded with AFM heparin-coated tip (medium panel) and corresponding histogram (right panel, obtained from 756 force curves) for ( A ) the wild-type strain of Msmeg , ( B ) Msmeg mc 2 155 expressing hbhA_Mtb , ( C ) the Δ MSMEG_1285 mutant and ( D ) the Δ MSMEG_1285 mutant expressing hbhA_Mtb . All the strains were cultured in 7H9 supplemented with OADC and without detergent. For each image, the red square corresponds to the area scanned for the determination of one adhesion map.

    Journal: International Journal of Molecular Sciences

    Article Title: Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria

    doi: 10.3390/ijms19061673

    Figure Lengend Snippet: Deletion of MSMEG_1285 in Msmeg mc 2 155 impacts the cell-surface exposure of HBHA_ Mtb . AFM tomographic image (left panel), representative spatially-resolved map of adhesion forces recorded with AFM heparin-coated tip (medium panel) and corresponding histogram (right panel, obtained from 756 force curves) for ( A ) the wild-type strain of Msmeg , ( B ) Msmeg mc 2 155 expressing hbhA_Mtb , ( C ) the Δ MSMEG_1285 mutant and ( D ) the Δ MSMEG_1285 mutant expressing hbhA_Mtb . All the strains were cultured in 7H9 supplemented with OADC and without detergent. For each image, the red square corresponds to the area scanned for the determination of one adhesion map.

    Article Snippet: Msmeg mc2 155, the deletion mutant Δ MSMEG_1285 and the corresponding complemented strain ( ) were grown in Sauton’s medium (containing 0.025% tyloxapol), in Middlebrook 7H9 broth or on 7H11 agar plates supplemented with OADC enrichment (Becton Dickinson, Le Pont-de-Claix, France), hygromycin (50 μg·mL−1 ) or kanamycin (25 μg·mL−1 ), when required.

    Techniques: Expressing, Mutagenesis, Cell Culture

    Heterologous expression of hbhA_Mtb in the Δ MSMEG_1285 mutant affects auto-aggregation and colony morphology. ( A ) Overnight cultures in 7H9 supplemented with OADC and without detergent of Msmeg mc 2 155 and the Δ MSMEG_1285 mutant, both expressing hbhA_Mtb ; ( B ) Serial dilution of Msmeg mc 2 155 (upper series) and the Δ MSMEG_1285 mutant (lower series), both expressing hbhA_Mtb , on 7H11 agar plates; ( C ) Western-blot analysis using monoclonal antibodies anti-Hsp65 (upper panel), anti-HBHA VF2 (medium panel) and anti-HBHA D2 (lower panel) on 40 µg of total lysates from Msmeg mc 2 155 and the Δ MSMEG_1285 mutant, both expressing hbhA_Mtb .

    Journal: International Journal of Molecular Sciences

    Article Title: Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria

    doi: 10.3390/ijms19061673

    Figure Lengend Snippet: Heterologous expression of hbhA_Mtb in the Δ MSMEG_1285 mutant affects auto-aggregation and colony morphology. ( A ) Overnight cultures in 7H9 supplemented with OADC and without detergent of Msmeg mc 2 155 and the Δ MSMEG_1285 mutant, both expressing hbhA_Mtb ; ( B ) Serial dilution of Msmeg mc 2 155 (upper series) and the Δ MSMEG_1285 mutant (lower series), both expressing hbhA_Mtb , on 7H11 agar plates; ( C ) Western-blot analysis using monoclonal antibodies anti-Hsp65 (upper panel), anti-HBHA VF2 (medium panel) and anti-HBHA D2 (lower panel) on 40 µg of total lysates from Msmeg mc 2 155 and the Δ MSMEG_1285 mutant, both expressing hbhA_Mtb .

    Article Snippet: Msmeg mc2 155, the deletion mutant Δ MSMEG_1285 and the corresponding complemented strain ( ) were grown in Sauton’s medium (containing 0.025% tyloxapol), in Middlebrook 7H9 broth or on 7H11 agar plates supplemented with OADC enrichment (Becton Dickinson, Le Pont-de-Claix, France), hygromycin (50 μg·mL−1 ) or kanamycin (25 μg·mL−1 ), when required.

    Techniques: Expressing, Mutagenesis, Serial Dilution, Western Blot

    Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo . Exponentially growing M. tuberculosis mc 2 6230 was incubated with increasing concentrations of CyC 17 or CyC 7β in 7H9 OADC/Tween 80 at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2- 14 C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A , chemical structures of the CyC analogs used in this study. B and C , effect of CyC 17 ( B ) or CyC 7β ( C ) treatment on the mycolic acid profiles of M. tuberculosis mc 2 6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( far left ). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) ( middle left ). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( middle right ). Densitometric analysis ( far right ) was performed on the TLCs shown in the left panels. Histograms and error bars , means and S.D. values calculated from at least two independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Cyclipostins and cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo

    doi: 10.1074/jbc.RA117.000760

    Figure Lengend Snippet: Ag85 complex mycolyltransferase activity is inhibited by CyC analogs in vivo . Exponentially growing M. tuberculosis mc 2 6230 was incubated with increasing concentrations of CyC 17 or CyC 7β in 7H9 OADC/Tween 80 at 37 °C with agitation for 1 h. Subsequently, bacteria were labeled with sodium [2- 14 C]acetate for 6 h at 37 °C with agitation. The cultures were split, and from the first volume were extracted the total methyl esters of mycolates (MAME) and fatty acids (FAME). From the second volume, apolar and polar lipid fractions were obtained before derivatization of arabinogalactan MAME. A , chemical structures of the CyC analogs used in this study. B and C , effect of CyC 17 ( B ) or CyC 7β ( C ) treatment on the mycolic acid profiles of M. tuberculosis mc 2 6230. Equal counts (50,000 cpm) of MAME + FAME fraction were loaded on a TLC plate and resolved once using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( far left ). The apolar fraction was loaded (50,000 cpm), and TMM and TDM were visualized on a 1D TLC plate using the solvent system chloroform/methanol/water (40:8:1, v/v/v) ( middle left ). Equal volumes of arabinogalactan MAME fraction were loaded, and α, methoxy, and keto mycolic acids were visualized on a 1D TLC plate using the solvent system hexane/ethyl acetate (95:5, v/v) run twice ( middle right ). Densitometric analysis ( far right ) was performed on the TLCs shown in the left panels. Histograms and error bars , means and S.D. values calculated from at least two independent experiments.

    Article Snippet: M. tuberculosis mc2 6230 ( ) was grown on Middlebrook 7H10 agar plates containing OADC (oleic acid, albumin, dextrose, catalase) enrichment (Difco) and supplemented with 24 μg/ml pantothenic acid.

    Techniques: Activity Assay, In Vivo, Incubation, Labeling, Thin Layer Chromatography

    M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in 7H9 culture medium supplemented with OADC and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).

    Journal: PLoS Pathogens

    Article Title: Impact of Mycobacterium ulcerans Biofilm on Transmissibility to Ecological Niches and Buruli Ulcer Pathogenesis

    doi: 10.1371/journal.ppat.0030062

    Figure Lengend Snippet: M. ulcerans ECM and Its Formation (A) Scanning electron micrographs of bacilli isolated by immunomagnetic beads separation from mammalian host tissues. Bacilli isolated from human (1) or mouse (2) tissue formed large clusters that are surrounded by ECM. Arrows indicate the immunomagnetic beads. Scale bar: 10 μm (1 and 2), 1 μm (inset). (B) Scanning electron micrographs of cluster formation by M. ulcerans . (1) Ten days after inoculation in 7H9 culture medium supplemented with OADC and containing Tween 80 0.05%, some bacilli formed a small cluster. (2) After 20 d, the bacilli had multiplied and the clusters were covered by extracellular material. (3) At the end of exponential growth (45 d after inoculation) a large cluster was formed (4), which was surrounded by the ECM. Scale bars: 1 μm (1), 1.5 μm (2), 25 μm (3), and 10 μm (4).

    Article Snippet: Then, 35 d later, exponentially growing bacteria from agar plates were harvested in 7H9 broth supplemented with 10% OADC and Tween 80 0.05% (Sigma) at 105 bacteria/mL.

    Techniques: Isolation

    Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto Middlebrook 7H10 plates containing OADC, glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.

    Journal: Molecular microbiology

    Article Title: NAD+ Auxotrophy is Bacteriocidal for the Tubercle Bacilli

    doi: 10.1111/j.1365-2958.2010.07099.x

    Figure Lengend Snippet: Growth of M. bovis Δ nadABC and M. tuberculosis Δ nadABC in vitro. A . The strains were grown with NAm (20 mg/l) to log phase, spun down, washed 5 X with PBS, resuspended in media without NAm, and inoculated into media containing either NAm at different concentrations or NAc at 20 mg/l. The complemented strains were grown without any supplement. The cultures were incubated with shaking at 37°C for 3 weeks and growth was followed by measuring OD 600nm . B . The strains were grown, washed, diluted, and inoculated in media containing or not containing NAm (20 mg/l). At each time point, samples were taken, diluted, and plated onto Middlebrook 7H10 plates containing OADC, glycerol, and NAm (20 mg/l). The plates were incubated at 37°C for 4-5 weeks and colonies were counted. The concentrations are in mg/l.

    Article Snippet: The strains were grown in Middlebrook 7H9 medium (Difco, Sparks, MD) supplemented with 10% (v/v) OADC enrichment (Difco), 0.2% (v/v) glycerol, and 0.05% (v/v) tyloxapol.

    Techniques: In Vitro, Incubation

    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p

    Journal: PLoS Pathogens

    Article Title: Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs

    doi: 10.1371/journal.ppat.1005949

    Figure Lengend Snippet: Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p

    Article Snippet: M . tuberculosis strains were grown in 7H10-OADC or Dubos-ADC media (Difco).

    Techniques: Derivative Assay, MTT Assay, Incubation, Labeling, Mutagenesis

    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in 7H9 OADC containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in 7H9 OADC containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques: Activity Assay, Recombinant

    Distribution of hit compound IC 50 values in macrophages and in 7H9 OADC. Dot plot depicting the IC 50 values for the most potent 1,359 compounds in 7H9 OADC and in the macrophage infection assays. For both assays, compounds were tested across 8 separate 2-fold dilution series 50–0.4 μM. Universally active compounds with IC 50 values

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: Distribution of hit compound IC 50 values in macrophages and in 7H9 OADC. Dot plot depicting the IC 50 values for the most potent 1,359 compounds in 7H9 OADC and in the macrophage infection assays. For both assays, compounds were tested across 8 separate 2-fold dilution series 50–0.4 μM. Universally active compounds with IC 50 values

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques: Infection

    Chemical rescue of Mtb ΔIcl1. (A) Growth of Mtb ΔIcl1 was monitored in 7H9 OADC containing cholesterol (100 μM) or propionate (100 μM) in the presence of V-13–012725 (25 μM) and V-13–011503 (25 μM). Growth rescue by the compounds V-13–012725 and V-13–011503 is specific to cholesterol with no growth is observed in media containing propionate. (B) The compound, V-13–009920 (25 μM) rescues Mtb ΔIcl1 growth in 7H9 OADC media containing cholesterol (100 μM) and propionate (100 μM). Chemical rescue by V-13–009920 is comparable to rescue by vitamin-B12 (10 μg/ml). The data are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: Chemical rescue of Mtb ΔIcl1. (A) Growth of Mtb ΔIcl1 was monitored in 7H9 OADC containing cholesterol (100 μM) or propionate (100 μM) in the presence of V-13–012725 (25 μM) and V-13–011503 (25 μM). Growth rescue by the compounds V-13–012725 and V-13–011503 is specific to cholesterol with no growth is observed in media containing propionate. (B) The compound, V-13–009920 (25 μM) rescues Mtb ΔIcl1 growth in 7H9 OADC media containing cholesterol (100 μM) and propionate (100 μM). Chemical rescue by V-13–009920 is comparable to rescue by vitamin-B12 (10 μg/ml). The data are representative of two independent experiments.

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques: