The M. smegmatis and M. tuberculosis was maintained on Middlebrook 7H9 broth containing 0.05% Tween 80 and 10% (v/v) OADC (Oleic Acid, Albumin, Dextrose, and Catalase) supplement. The culture screening was performed by Ziehl–Neelsen staining before used in the antimicrobial assays. Two-fold serial dilutions of CLCLE extract and Rifampicin were made with 100 μl of each; sterile distilled water and 7H9 Middlebrook culture medium for M. tuberculosis and M. smegmatis in plates of 96 well microplates. The plates were incubated at 37°C for 24 days. The developer (40 μL) used was iodonitrotetrazolium (INT) of Sigma–Aldrich. Minimum Inhibitory Concentration (MIC) values were recorded as the lowest concentrations of extracts showing no growth, and bacterial growth in the wells was indicated by color change (Damato and Collins, 1990 (link); Sadaphal et al., 2008 (link)).
Iodonitrotetrazolium int
Manufactured by Merck Group
Sourced in United States
Iodonitrotetrazolium (INT) is a colorimetric redox indicator used in biochemical and microbiological applications. It functions as an electron acceptor, undergoing reduction to produce a colored formazan product. INT is commonly utilized in assays to detect and quantify cellular metabolic activity and viability.
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Lab products found in correlation
3 protocols using iodonitrotetrazolium int
1
Antimicrobial Assay of CLCLE Extract
2
Eag1 Knockdown Affects Cell Proliferation
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Cell lines cultured in logarithmic growth phase were inoculated in 96-well plates. After culturing for 12 hours with serum-free 1640 medium, the GC cells were transfected with Eag1-shRNA and Eag1-shRNA-NC (empty vector). The transfected cells were mixed with 10 µL of cholecystokinin octapeptide (CCK-8; Biosharp, Hefei, China) solution and incubated for another 4 hours at 37 ℃. A microplate reader with the absorbance of 450 nm was used for cell detection.
Clone formation was performed in transfected cells (Eag1-shRNA and empty vector), which were cultured for 3 weeks at 37 ℃ in the presence of 5% CO2. After dropping the culture solution and fixing with 4% methanol for 15 minutes, the plates were stained with iodonitrotetrazolium (INT; Sigma, St. Louis, MO, USA). The number of colonies was counted by microscopic observation. Cloning efficiency was defined as the number of cell colonies/inoculated cell number ×100%.
Clone formation was performed in transfected cells (Eag1-shRNA and empty vector), which were cultured for 3 weeks at 37 ℃ in the presence of 5% CO2. After dropping the culture solution and fixing with 4% methanol for 15 minutes, the plates were stained with iodonitrotetrazolium (INT; Sigma, St. Louis, MO, USA). The number of colonies was counted by microscopic observation. Cloning efficiency was defined as the number of cell colonies/inoculated cell number ×100%.
3
Microdilution Antimicrobial Susceptibility Test
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The microdilution method was used to determine the minimum inhibitory concentration (MIC) value. Each of the extracts and chloramphenicol (positive control) were dissolved in DMSO to obtain final concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 mg/mL, respectively. In addition to positive and negative controls, a total of 100 µL of each concentration was placed into 96 microplate wells. Each well was then inoculated with a bacterial suspension of 5 µL and incubated at 37 o C. Tests were performed in triplicate. After 18 hours, 20 µL of iodonitrotetrazolium (INT, Sigma Aldrich) solution was added to each microplate well and incubated again for 30 minutes at 37 o C. Bacterial growth was indicated by a color change in the wells, namely from pink to red. MIC values were determined by identifying the weakest concentration that did not exhibit bacterial growth [11] .
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