Complete Freund’s adjuvant was purchased from InvivoGen, San Diego, CA, USA (vac-cfa-10). Methotrexate (≥98% (HPLC), A6770); silibinin (containing both A and B diastereomers, ≥98% (HPLC), S0417); DTNB (≥98%, D-8130); NADPH (≥97% (HPLC), N-7505); glutathione (GSH ≥ 98.0%, G4251); glutathione reductase; polyvinylpolypyrrolidone (PVPP, 77627); and nitrotetrazolium bluechloride (NBT, ≥90.0% (HPLC), 298-83-9) were purchased from (Sigma-Aldrich Co, St. Louis, MA, USA). Alanine aminotransferase (ALT) kits were purchased from Analyticon ® Biotechnologies AG, Frankfurt, Germany. Aspartate aminotransferase (AST) kits were purchased from Spinreact, S.A./S.A. U Ctra. Santa Coloma, Gerona, Spain. Trichloroacetic (TCA); thiobarbituric acid (TBA); hydrogen peroxide, (H2O2 30% w/v); L-methionine (149 g/mol); riboflavin (376 g/mol); potassium dihydrogen orthophosphate (KH2PO4); dipotassium hydrogen orthophosphate (K2HPO4); and EDTA sodium salt were made available by the Beirut Arab University.
Nitrotetrazolium blue chloride nbt
Manufactured by Merck Group
Sourced in United States
Nitrotetrazolium Blue chloride (NBT) is a chemical compound commonly used as a histochemical stain and redox indicator in various laboratory applications. It functions as a soluble tetrazolium salt that is reduced to an insoluble, blue-colored formazan precipitate in the presence of enzymatic activity or reducing agents. The core function of NBT is to facilitate the visualization and detection of such enzymatic or redox-related processes in biological samples.
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Lab products found in correlation
Noble agar, by Thermo Fisher Scientific (2 mentions)
Cell death detection elisaplus, by Merck Group (2 mentions)
5 bromo 4 chloro 3 indolyl phosphate bcip, by Merck Group (2 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
Methotrexate, by Merck Group (1 mentions)
Silibinin, by Merck Group (1 mentions)
Complete freund s adjuvant, by InvivoGen (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Stemi sv6, by Zeiss (1 mentions)
Difco noble agar, by BD (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
7 protocols using nitrotetrazolium blue chloride nbt
1
Freund's Adjuvant-Induced Oxidative Stress Assay
2
Histochemical Staining of Plant Leaves
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Mock-, BaMV-, and BaMV△CPN35-ILs of N. benthamiana at 10 dpi or of B. distachyon at 7 dpi were detached for histochemical staining as described previously with minor modification (Fryer et al., 2003 (link)). Briefly, 0.2% (w/v) Nitrotetrazolium blue chloride (NBT) (Sigma-Aldrich) in 50 mM sodium phosphate buffer (pH 7.5) and 0.2% (w/v) DAB (Sigma-Aldrich) (pH 3.8) were used for superoxide and H2O2 detection. The detached leaves were immediately immersed in freshly prepared NBT or DAB staining buffer followed by vacuum infiltration for 10 min, before being kept in the dark overnight. Then, the leaves were washed once with distilled water and repeatedly shaken in 95% (v/v) alcohol for 20 min until the green color of chlorophyll had disappeared.
3
Quantifying Anchorage-Independent Growth
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DNA fragmentation was assayed using a Cell Death Detection ELISA PLUS (Sigma Aldrich, St. Louis, MO). Anchorage-independent growth (AIG) was assayed as described previously (9 (link)). For each cell line, 1 × 104 cells were suspended in a top layer of 0.36% noble agar (USB Corporation) and a bottom layer of 0.4% agar both in RPMI-1640 + 10% FBS in a 6-well culture dish. After 1–3 weeks, colonies were stained with Nitrotetrazolium Blue chloride (NBT; Sigma) and photographed with a S600 Canon camera. Colonies were quantified with ImageJ analysis software.
4
Histochemical Staining of hiPSCs
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The hiPSCs were fixed in 4% paraformaldehyde in PBS for 5 min, and then incubated in 0.1% Triton X-100 in PBS for 5 min at room temperature (RT). After rinse with NTMT substrate buffer (0.1 M Tris-HCl, pH 9.5 containing 0.1 M NaCl, 5 mM MgCl2 and 0.1% Triton X-100), the cells were incubated in fresh NTMT buffer containing 0.175 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma-Aldrich) and 0.45 mg/mL of nitrotetrazolium blue chloride (NBT; Sigma-Aldrich) for 30 min at RT. Samples were then washed two times with PBS and observed under an inverted light microscope.
5
Quantifying Anchorage-Independent Growth
6
Histochemical Evaluation of hPLAP Expression
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Histochemical staining was performed to evaluate hPLAP expression as described40 (link). In brief, cells were fixed in ice-cold acetone–methanol (30:70, v/v) for 5 min. Following two washing steps using PBS, endogenous heat-labile alkaline phosphatases were inactivated by incubation in TMN substrate buffer (0.1 M Tris- HCl, pH 9.5 containing 0.1 M NaCl and 5 mM MgCl2) at 60 °C for 30 min. Thereafter, staining for heat-stable hPLAP was performed at RT for at least 3 h by using fresh TMN buffer containing 0.175 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma-Aldrich) and 0.45 mg/mL of nitrotetrazolium blue chloride (NBT; Sigma-Aldrich)40 (link). After two washing steps, samples were evaluated under a stereo microscope (Stemi SV6; Zeiss) or an inverted light microscope (Axiovert 25; Zeiss). Each sample was stained at least in triplicate. MSC isolated from WT mice served as negative control.
7
Soft Agar Colony Formation Assay
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Soft agar (1% w/v Difco agar noble (Becton Dickinson) in deionized water) was mixed with an equal volume of 2X complete RPMI1640 medium at 40 °C. 320 μl of agar mix was added to each well of a 24-well culture plate and allowed to solidify for 30 min at RT. Cells were re-suspended in an equal volume of 0.6% soft agar mix at 40 °C to yield a final concentration of 4000 cells/ml. The cell-agar mix was layered on top of the solidified agar mix and allowed to solidify for 30 min at RT. Complete RPMI1640 medium was added on top of the agar to prevent desiccation and cells were grown for 14 days. Cell colonies were examined by microscope under a 4X objective with phase contrast (Olympus IX83 microscope, Japan). Cells were also stained with nitrotetrazolium blue chloride (NBT) (Sigma-Aldrich) overnight under culture conditions for clearer visualization of colonies. The area of tumor colony was determined using the CellSens Dimension software (Olympus).
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