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Determination of Nitric Oxide (NO) and cytokine levels

NC Nimsi Campos-Xolalpa
ÁA Ángel Josabad Alonso-Castro
EO Elizabeth Ortíz-Sanchez
JZ Juan Ramon Zapata-Morales
MG Marco Martin González-Chávez
SP Salud Pérez
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In 6 well-plates, 5 × 105 macrophages J774A.1 were seeded per well. Macrophages were stimulated with 5 μg/mL LPS. After 2 h, cells were treated with ECL (10 μg/mL), DAM and ICT (25 μg/mL), DEOX (20 μg/mL), or reference drug (IND 17.1 μg/mL), and (culture medium with vehicle only). After 24 h, supernatants were collected for the quantification of NO and levels of IL-1β, IL-6, IL-10, and TNF-α, using commercial enzyme-linked immunosorbent assays (ELISA), following the manufacturer’s instructions (PROMEGA). The absorbance was recorded at 405 nm. For the quantitation of NO production, 100 μL of supernatant was mixed with 100 μL of Griess reagent in a 96-well plate. The reaction mixture was incubated at 37 °C for 30 min and the absorbance was measured at 540 nm with an ELISA reader (Bio-Rad). A 100% nitric oxide production was considered for the LPS group [15].

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