Essay on Assay And The Cell Viability

1159 Words Dec 7th, 2016 5 Pages
A colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemical Co.) assay was performed to measure the cell viability. Briefly, RAW 264.7 cells were treated with different concentrations of spermidine for 24 h or pretreated with spermidine for 1 h before stimulation with LPS for 24 h. After incubation, the medium was discarded, and MTT solution (5 mg/mL in phosphate-buffered saline, PBS) was added to each well and incubated for another 3 h at 37 °C. The medium was removed, and DMSO was added to dissolve the formazan dye. The optical density was then read at 560 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) to determine the cell viability.

Measurement of NO production in RAW 264.7 macrophages
The production of NO in the culture supernatants was assayed using Griess reagent (Sigma-Aldrich Chemical Co.). For this assay, the supernatant was collected and mixed with the same volume of Griess reagent for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using a microplate reader, and NO concentrations were calculated by referencing a standard curve generated from the known concentrations of sodium nitrite [Lee et al., 2015].

Measurement of PGE2, TNF-α, and IL-1β production in RAW 264.7 macrophages
To measure the production of PGE2, TNF-α, and IL-1β, the cells were cultured under the same conditions as for the NO measurement assay. The levels of PGE2, TNF-α, and IL-1β…

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