Microsep device

The ELISA kit (DUO Set, R&D systems, Abingdon, UK) was used as per manufacturer's instructions, as described previously, to monitor the TNF-alpha release from murine macrophages (5) (link). Apa1 monomer (55 kDa), oligomeric form of apa1 (350 kDa), apa2, royal jelly peptide apisimin, 1 % (w/v) honey solution, proteins purified from the honeys, and deproteinised honey samples prepared in complete RPMI 1640 medium were applied in the stimulation assay. Lipopolysaccharides of Salmonella typhimurium (LPS) were used as a positive control. The proteins were separated from honey by filtration using a Microsep device (Pall Life Sciences, Ann Arbor, MI, USA) with 3 kDa cut-off. Deproteinised honey solution [30 % (w/v)] was adjusted to 1 % (v/v) in complete RPMI 1640 medium. All prepared honey solutions were rendered sterile by membrane filtration (0.22 µm). The level of TNF-α was determined in cell culture supernatants collected after 3, 6, and 24 h of cultivation. A recombinant mouse TNF-α was used as a standard. The assay was repeated three times.