Measurement of nitrite concentration was done using the Griess Reaction Assay (13 ) . Nitrite oxide (NO) released into the supernatants of cultured macrophages and is unstable and rapidly converts to nitrite and nitrate. Detection of nitrite amount as an estimation of NO level synthesis in the cultures was determined by the standard Griess reagent [1% sulfanilamide/0.1% N-(1-naphthyl) ethylenediamine dihydrochloride/2.5% H3PO4] (Merck). In brief, 50 μl of test solution (supernatants of macrophage culture) mixed with 50 μL of Griess reagent in a 96-well flat-bottomed plate in triplicate order. After 15 min, absorbance was measured in a Multiskan MS microplate reader at 540 nm. Nitrite concentration was determined from a standard curve of sodium nitrite (14 (link)).
Ms microplate reader
Manufactured by MultiSciences Biotech
The MS microplate reader is a versatile laboratory instrument designed to measure the absorbance, fluorescence, or luminescence of samples in microplates. It can be used for a variety of applications, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other microplate-based experiments.
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2 protocols using ms microplate reader
1
Nitrite Quantification using Griess Assay
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Measurement of nitrite concentration was done using the Griess Reaction Assay (13 ) . Nitrite oxide (NO) released into the supernatants of cultured macrophages and is unstable and rapidly converts to nitrite and nitrate. Detection of nitrite amount as an estimation of NO level synthesis in the cultures was determined by the standard Griess reagent [1% sulfanilamide/0.1% N-(1-naphthyl) ethylenediamine dihydrochloride/2.5% H3PO4] (Merck). In brief, 50 μl of test solution (supernatants of macrophage culture) mixed with 50 μL of Griess reagent in a 96-well flat-bottomed plate in triplicate order. After 15 min, absorbance was measured in a Multiskan MS microplate reader at 540 nm. Nitrite concentration was determined from a standard curve of sodium nitrite (14 (link)).
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Measurement of nitrite concentration was done using the Griess Reaction Assay (13 ) . Nitrite oxide (NO) released into the supernatants of cultured macrophages and is unstable and rapidly converts to nitrite and nitrate. Detection of nitrite amount as an estimation of NO level synthesis in the cultures was determined by the standard Griess reagent [1% sulfanilamide/0.1% N-(1-naphthyl) ethylenediamine dihydrochloride/2.5% H3PO4] (Merck). In brief, 50 μl of test solution (supernatants of macrophage culture) mixed with 50 μL of Griess reagent in a 96-well flat-bottomed plate in triplicate order. After 15 min, absorbance was measured in a Multiskan MS microplate reader at 540 nm. Nitrite concentration was determined from a standard curve of sodium nitrite (14 (link)).
2
Croton tiglium Extract Cytotoxicity on A549 Cells
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In logarithmic phase, A549 cells were made into mono-cellular suspension and seeded in a 96-well plate with RPMI 1640 medium (supplemented with 10% FBS) at a density of 1×104 cells/well for totally 200 μl for a 24 h incubation. The cells were treated with Croton tiglium extract with a series of concentrations (0, 10, 50, 100 and 200 μg/ml) and cultured in 2% FBS medium for 12, 24, and 48 h. The morphology of cells were observed and photographed under the inverted microscope (×100). 20 μl 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT, 5 mg/ml, Sigma, USA) were added to each well and incubated for an additional 4 h. Then, the medium was removed and 150 μl DMSO were added to each well. The absorbance (A) was measured at A490 nm by a Multiskan MS microplate reader. All experiments were conducted in parallel with vehicle controls (0.1% DMSO). Four parallel holes were established in each group and the study was repeated for 3 times to obtain the mean values. Cell survival rate (%) = (treatment group absorbance/control group absorbance) × 100%. The IC 50 value was defined as a concentration of compound that reduces absorbance by 50% in comparison with vehicle-treated well and five concentrations were applied to determine an IC 50 value of Croton tiglium extract on A549.
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