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SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 115diphenyl-tetrazolium bromide (MTT), dimethyl sulphoxide (DMSO) (Sigma), along with other necessary chemicals [10].Method for Determining Nitric Oxide (NO) Production Inhibition CapacityCell Seeding: RAW 264.7 cells were seeded at a density of 2 %u00d7 105 cells per well in a 96-well plate and incubated at 37%u00b0C with 5% CO2 for 24 hours.Media Replacement: After incubation, the culture medium was replaced with serum-free DMEM for 3 hours.Treatment: Cells were then treated with the test samples at varying concentrations for 2 hours, followed by stimulation with LPS (10 %u03bcg/mL) for 24 hours to induce NO production.Nitrite Measurement: Nitrite (NO2-) levels, used as an indicator of NO production, were measured with the Griess reagent. A 100 %u03bcL aliquot of the culture medium was transferred to a new 96-well plate and mixed with 100 %u03bcL of Griess reagent, incubated at room temperature for 10 minutes. Absorbance was read at 540 nm. A well with culture medium alone served as the negative control, and wells with LPS-stimulated cells served as the positive control. L-NMMA was used as a reference inhibitor [11].Data Analysis: Nitrite concentrations were calculated using a standard curve prepared with NaNO2 and were compared as a percentage against the positive control. NO inhibition (%) was calculated using the following formula: %NOinhibition = (ODpositive control (+) %u2013 ODsample)/(ODpositive control (+) %u2013 ODnegative control(-)) x 100%Replicates and IC50 Calculation: Experiments were conducted in triplicate, and IC50 (the concentration required to inhibit 50% NO production) was determined using Microsoft Excel.