Griess reagent assay

NO₂⁻ levels in liver and cell culture supernatant were measured using the Griess reagent assay according to the manufacturer (Promega GmbH, Madison, WI, USA) and normalized to liver protein concentration.

Macrophages were stimulated with 100 ng/mL LPS for 12 hours, and the supernatant (SN) was collected, filtered through a 0.45 μm filter, and applied to rMAPC for 12 hours. Supernatant of untreated macrophages was used as a control. To define differences in messenger ribonucleic acid (mRNA) expression, rMAPC were incubated with SN of ±LPS-activated macrophages or LPS (7.5 × 10⁵ cells/well). Exposure to the SN of LPS-activated macrophages was prolonged to 18 hours for measuring NO using the Griess reagent assay (Promega, Leuven, Belgium).
To prepare double-conditioned media (DCM), rMAPC (5 × 10⁴ cells/well), previously exposed to the SN of LPS-activated macrophages for 12 hours, were allowed to secrete soluble factors for 24 hours in 50 μL macrophage medium (96-well plate). In specific experiments, rMAPC were stimulated with a mixture of recombinant rat TNF-α, IL-1β, and IL-6 (100 ng/mL each; all from Peprotech, London, UK) to partially mimic the SN of LPS-activated macrophages. This conditioned medium is designated as “licensed-” conditioned medium (LCM). Nonstimulated rMAPC provided the single-conditioned media (CM). The representation of the generation of conditioned media from rMAPC is illustrated in Supplemental Figure 1 available online at https://doi.org/10.1155/2017/2353240. All the conditioned media were collected and filtered through a 0.45 μm filter.

Arginase activity in liver tissue was assessed using the modified method described by Corraliza et al. [32 (link)] and detailed previously [33 (link)]. MPO activity was determined in homogenized liver tissue as previously detailed [34 (link)] adapted to mice hepatic tissue (~50 mg tissue homogenized in 300 μL Dulbecco’s Phosphate Buffered Saline (DPBS, PAN Biotech, Aidenbach, Germany)), and normalized to protein concentration. Nitric oxide (NOx) concentration was measured in the cell supernatant using the Griess reagent assay (Promega, Mannheim, Germany).

The amount of nitrite in the culture medium was measured using Griess reagent as a substitute for NO production by BV-2 cells. BV-2 cells were transferred onto 96-well plates (1 × 10⁵ cells/well) and incubated for 2 h at 37 °C, 5% CO₂. Cells were activated by 3 ng/mL LPS and up to 80 U/mL IFN-γ for 24 h at 37 °C, 5% CO₂. 50 μL of cell culture medium was used for Griess Reagent Assay (Promega, Madison, WI, USA), as described in the manufacturer’s protocol. Experiments were conducted in triplicate and repeated at least 3 times.

Cell supernatants were used for quantification of cytokines (IL1-β, IL-6 and TNF-α) by commercial ELISA Kits following the protocols provided by the manufacturer (KEY RESOURCES table). To measure nitrite concentrations, cell culture supernatants were collected and immediately subjected to a Griess Reagent assay (Promega), according to the suppliers’ protocol.
Serum anti-dsDNA-IgG levels were measured at week 5, 10, 16, 20 and 24 using an ELISA Kit (Alpha Diagnostic International) according to the manufacturer’s instructions.