1 × 103 JJN3 cells or 5 × 103 U266 cells were plated in 1 mL of Methocult™ H4230 methylcellulose semi-solid media (Stem Cell Technologies™, Cambridge, UK) and treated with 1 µL of DMSO or Olaparib stock to achieve desired concentration. Plates were left in a humidified incubator at 37°C for 10 days and colonies were stained with Iodonitrotetrazolium Chloride (INT; Sigma Aldrich) at a concentration of 8 mg/mL. Colonies were counted using a light microscope.
Iodonitrotetrazolium chloride int
Manufactured by Merck Group
Sourced in United States
Iodonitrotetrazolium chloride (INT) is a chemical compound commonly used in biochemical and microbiological applications as an indicator. INT functions as a colorimetric redox indicator, undergoing reduction to a colored formazan compound in the presence of electron transport systems or dehydrogenase enzymes.
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Lab products found in correlation
Methocult h4230, by STEMCELL (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (1 mentions)
Ethanol, by AppliChem (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Ethylenediaminetetraacetic acid edta, by Carl Roth (1 mentions)
Prism 9, by GraphPad (1 mentions)
Triethanolamine, by Merck Group (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane, by Merck Group (1 mentions)
Uv 1800, by Shimadzu (1 mentions)
6 protocols using iodonitrotetrazolium chloride int
1
Clonogenic Assay for Myeloma Cells
2
Antibacterial Activity Determination
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Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591) and Pseudomonas aeruginosa (ATCC 27853) strains were used for the antibacterial assay. The minimum inhibitory concentration (MIC) of the extract and the isolated compounds were determined in triplicate according to Wiegand et al. (2008) (link) in Mueller–Hinton medium (MH). After the incubation of the inoculated 96-well plates at 37°C for 24 h, iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was added to each well at a final concentration of 0.2 mg/ml and incubated for 20 min (Eloff, 1998 (link)). The highest dilution of a compound in which no growth appears corresponds to its MIC. Gentamicin for P. aeruginosa and chloramphenicol for S. aureus were used as controls.
3
Antibacterial Activity of Compounds
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Pseudomonas aeruginosa (ATCC 27853) was used for the antibacterial assay. The MIC of the different compounds was determined in triplicate using the broth dilution method in 96-well microtiter plates [52 (link)]. Briefly, compounds were resuspended at 10.24 mg/mL in DMSO and serially diluted in Mueller−Hinton broth (MHB, Oxoid). The maximum initial concentration used for this assay was 256 μg/mL. After an incubation of 24 h at 37 °C, iodonitrotetrazolium chloride (INT, Sigma-Aldrich, St Louis, MO, USA) was added to each well, as growth indicator, and incubated for 1 h [53 (link)]. The highest dilution of a compound in which no growth appeared corresponded to its MIC. Gentamicin (INT, Sigma-Aldrich, St Louis, MO, USA) was used as control of inhibition and compared to the reference values.
4
Colorimetric Lactate Assay for Cell Cultures
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Lactate concentrations in supernatants of cultured cells were measured by a colorimetric assay, in which lactate is converted into pyruvate under the consumption of nicotinamide adenine dinucleotide (NAD) (Babson and Phillips 1965 (link); Limonciel et al. 2011 (link)). Reactive solutions were prepared on the basis of Limonciel et al. (2011 (link)). Triethanolamine (Sigma), ethylene diamine tetraacetic acid (EDTA; Roth), magnesium chloride (Applichem), N-methylphenazinium methosulfate (PMS; Sigma), iodonitrotetrazolium chloride (INT; Sigma), ethanol, Triton®X-100 (AppliChem), ß-nicotinamide-adenine-dinucleotide hydrate (NAD; Sigma) and lactate dehydrogenase (LDH; Sigma) were combined for the color solution. 10 µL of supernatant was mixed with 90 µL of color solution. For quantification, a standard curve with sodium-l -lactate (Sigma) from 50 to 0.2 mM was generated and lactate concentrations in supernatants were interpolated using non-linear-curve-fitting by GraphPad Prism 9.1.2 (GraphPad Software).
5
Purification and Characterization of Mulberry Flavonoids
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Morin, morusin, mulberrin, and morusinol were all purchased from Weikeqi Biotechnology Company (Chengdu, Sichuan, China). Cyclomorusin and cyclomulberrin were bought from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China) . These flavonoid compounds were all extracted from mulberry trees with 75% EtOH and further purified from the ethyl acetate extract.
The purities of these flavonoid compounds were all above 95%. Iodonitrotetrazolium chloride (INT) and ampicillin were both purchased from Sigma-Aldrich (St. Louis, MO, USA). Nutrient broth was purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. (Guangzhou, Guangdong, China).
The purities of these flavonoid compounds were all above 95%. Iodonitrotetrazolium chloride (INT) and ampicillin were both purchased from Sigma-Aldrich (St. Louis, MO, USA). Nutrient broth was purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. (Guangzhou, Guangdong, China).
6
Soil Dehydrogenase Activity Assay
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The enzyme activity of the tested soils was measured by the dehydrogenase activity according to the 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride (INT) assay (Von Mersi and Schinner, 1991) . In brief, each soil sample was mixed with Tris Buffer (Tris(hydroxymethyl)aminomethane, 1 M, Sigma-Aldrich) and substrate solution (2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (iodonitrotetrazolium chloride (INT), 10 mM, Sigma-Aldrich), and then incubated in the dark for 2 h at 40 °C. After the incubation, the soil was extracted by a solution consisting of N,Ndimethylformamide/ethanol in a 1:1 ratio, in the dark for 30 min at 20 °C to extract the developed iodonitrotetrazolium formazan (INTF). The developed INTF was determined color metrically at λ = 464 nm (UV-1800, Shimadzu, Kyoto, Japan). The dehydrogenase activity was expressed as mg INTF/g dry soil /2 h.
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