Nitro bt

Manufactured by Merck Group

Nitro-BT is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information about the core function and intended use of this product is not available.

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Lab products found in correlation

Cyquant cell proliferation assay, by Thermo Fisher Scientific (2 mentions) Cyquant assay, by Thermo Fisher Scientific (1 mentions) Polyvinyl alcohol (pva), by Merck Group (1 mentions) Adenosine diphosphate (adp), by Roche (1 mentions) Glutamate, by Merck Group (1 mentions) Cyquant assay kit, by Thermo Fisher Scientific (1 mentions)

4 protocols using nitro bt

Anchorage-dependent and Independent Proliferation Assay

Cited 3 times

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Anchorage dependent and independent proliferation was measured as previously described [3 (link), 4 (link), 34 (link)]. Anchorage-independent growth was assessed by plating cells in 0.4% agar in triplicate. Briefly, H358, H2122 and A549 cells with or without AGL expression were plated (15,000 cells/well) in triplicate in 6 welled dish. Colonies were stained with Nitro-BT (Sigma) at the end of the experiment and counted using Image J.
For anchorage dependent growth assay, cells with or without AGL expression were transfected with control siRNA or siRNA targeting HAS2 or RHAMM [5 (link)]. 72hrs after transfection cell proliferation and viability was assessed by plating 10³ cells per well in 96-well plates in triplicate for proliferation studies. CyQUANT® Cell Proliferation Assay (Invitrogen) was carried out according to manufacturer instructions to measure cell proliferation. To determine the effects of 4MU on cell viability, cells were plated as described, and treated with 4MU (600 μM) or 4MU (600μM) and HA(20μg/ml) together for 5 days. Cell viability was determined by CyQUANT Assay (Invitrogen).

Richmond C.S., Oldenburg D., Dancik G., Meier D.R., Weinhaus B., Theodorescu D, & Guin S. (2018). Glycogen debranching enzyme (AGL) is a novel regulator of non-small cell lung cancer growth. Oncotarget, 9(24), 16718-16730.

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Measuring Glutamate Dehydrogenase Activity

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To measure PAG activity, 41 U GDH (Serva Electrophoresis, GmbH; Heidelberg, Germany), dissolved in 50% glycerol, was placed on glass slides and equally distributed by spreading the solution across the glass slide with a coverglass. The glass slides were dried at room temperature in a vacuum (<1^-6 bar). Afterwards, 7-µm-thick cryostat tissue sections were cut (for lung tissues, 8-µm-thick sections were cut), captured onto GDH-coated glass slides, and stored at -80C. Before incubation, tissue sections were air dried at room temperature for 30 min.
In order to ensure excess GDH activity and its even spreading over the glass, GDH activity was measured by incubating the GDH films after pouring on top of the films incubation medium containing 18% PVA (Sigma-Aldrich; St. Louis, MO), 0.1 M phosphate buffer (pH 8.0), 5 mM NitroBT (Sigma-Aldrich), 3 mM NAD⁺ (Roche Applied Science; Basel, Switzerland), 2 mM ADP (Roche Applied Science), and 0.32 mM PMS (Serva Electrophoresis, GmbH) in the presence of 10 mM glutamate (Merck; Darmstadt, Germany). NitroBT was dissolved by heating NitroBT in a 1:1 solution of 100% ethanol and dimethylformamide with a final concentration of 2% v/v (0.34 M ethanol and 0.26 M dimethylformamide) for each solvent.

Botman D., Tigchelaar W, & Van Noorden C.J. (2014). Determination of Phosphate-activated Glutaminase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry). Journal of Histochemistry and Cytochemistry, 62(11), 813-826.

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Cell Proliferation and Soft Agar Assays

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For proliferation assay, cells were plated onto 96-well plates and allowed to grow for 5 days with freezing one plate each day. All plates were then processed with CyQuant assay kit (Life Technologies). For soft agar assays, 20,000 cells were suspendedin 0.4% agar and plated on 1% agar hard base in each well of 6-well plate. Colonies formed after 9–18 days in culture were stained with Nitro-BT (Sigma-Aldrich) at 37°C and counted using software ImageJ.

Agarwal N., Dancik G.M., Goodspeed A., Costello J.C., Owens C., Duex J.E, & Theodorescu D. (2016). GON4L drives cancer growth through a YY1-androgen receptor-CD24 axis. Cancer research, 76(17), 5175-5185.

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Anchorage-Dependent and Independent Cell Proliferation

Cited 1 time

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Anchorage dependent and independent proliferation was measured as before [4 (link), 9 (link)]. Briefly, shCTL and shAGL bladder cancer cells were transfected with control siRNA or siRNA targeting CD44 or RHAMM. 72 h after transfection anchorage-independent growth was assessed by plating cells in 0.4 % agar in triplicate. Colonies were stained with Nitro-BT (Sigma) and counted using Image J. Cell proliferation and viability was assessed by plating 10³ cells per well in 96-well plates in triplicate for proliferation studies. CyQUANT® Cell Proliferation Assay (Thermo Fisher Scientific) was carried out according to manufacturer instruction to measure cell proliferation.

Oldenburg D., Ru Y., Weinhaus B., Cash S., Theodorescu D, & Guin S. (2016). CD44 and RHAMM are essential for rapid growth of bladder cancer driven by loss of Glycogen Debranching Enzyme (AGL). BMC Cancer, 16(1), 713.

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