J774.2 cells (5 × 105) were incubated with native, dissociated, and N-deglycosylated hemocyanins (500 μg/ml) in serum-free culture medium for 24 h at 37 °C as described by Jiménez et al. (30 (link)). As a positive control, cells were incubated with LPS from E. coli (Enzo Life Sciences) (100 ng/ml). As a negative control, cells were incubated with culture medium without hemocyanin. Culture supernatants were collected and quantified by ELISA using BD OptEIATM kits (BD Biosciences) to detect IL-6, IL-12p40, and TNFα, according to the manufacturer's instructions.
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