*2.5. Capturing of TNF-α Present in Conditioned Medium of Macrophage Culture*

A human monocytic cell line THP-1 was maintained in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 μg/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES, and 10% fetal bovine serum (complete RPMI, cRPMI) (Life Technologies, Carlsbad, CA, USA). For the induction of cell differentiation, cells (106 per mL) were seeded in cRPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) for 24 h. After incubation, non-attached cells were removed by aspiration, and the adherent cells were washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype before stimulation, cells were incubated for an additional 48 h in cRPMI without PMA. For stimulation and retrieval of conditioned media, cells were further incubated for 4 h with 100 ng/mL of lipopolysaccharide (LPS) in fresh cRPMI and the supernatants

were collected and stored at −80 ◦C. Production of TNF-α was assessed in the conditioned media by commercial ELISA (Life Technologies, Carlsbad, CA, USA). Four systems were tested at least in quadruplicates, in two independent assays: (1) the biofunctionalized NFM (with immobilized TNF-α antibody); (2) a positive control with the soluble TNF-α antibody; (3) a negative control with the UV-O activated NFM; and (4) the conditioned cell culture medium alone. Each system was placed in sterile Eppendorf tubes and 2 mL of monocyte-derived macrophage conditioned medium was added individually, and kept at 37 ◦C in agitation in an orbital shaker at 120 rpm. Initially, a 3 days assay was performed with a 1 ng/mL TNF-α concentration from monocyte-derived macrophage conditioned medium. A specimen was collected at the following time points (2, 4, 6, 8, 10, 12, 24, 32, 48, and 72 h of incubation) without conditioned medium replacement. Afterwards, a 15-day assay was conducted, where a sample was collected daily and recharged with the same volume of recovered cell culture medium each day, which had a TNF-α concentration of 500 pg/mL. This concentration was chosen due to being closer to TNF-α levels in active RA patients (76.1 ± 103.2 pg/mL) [19], considering a margin of error affected by the TNF-α degradation.
