Determination of cytokines
The concentration of TNF-α, IFN-γ, IL-1β, IL-4, IL-6 and IL-10 cytokines
from the supernatants was determined by commercial Enzyme-linked
immunosorbent assay kits (ELISA) (Peprotech, Inc.) according to the
manufacturer ̵́s instructions. Briefly, plates were coated with either
TNF-α, IFN-γ, IL-1β, IL-4, IL-6, or IL-10 antibodies. Incubation with
the specific biotin-conjugated antibody, avidin-HRP conjugate, and
peroxidase chromogenic substrate were performed. Absorbance was measured
at 490 nm using a microplate absorbance reader (Thermo Scientific,
Multiskan FC). Cytokine concentration in supernatants was calculated by
the respective standard curves (0-2000 pg/ml). Macrophages stimulated
with lipopolysaccharide (LPS, 1 µg/mL) or methotrexate (MTX, 10 µg/mL)
were used as positive controls depending on whether the assay pretend to
stimulate (pro- or anti-inflammatory cytokines). Non-stimulated
macrophages (only supplemented DMEM) were used as basal control.
Afterwards, supernatants were collected and stored at -80ºC until
further analysis. All experimental variants were performed in
triplicate.