2.3.1. Monocyte Activation Test (MAT)

All studies involving human cells were conducted in accordance with the guidelines of the World Medical Association's Declaration of Helsinki. According to the national regulations, no additional approval by the local ethics committee was required in the case of anonymous blood cells discarded after plasmapheresis (buffy coats). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of anonymous donors by density gradient centrifugation. From the PBMCs, monocytes were then purified using magnetically-activated cell sorting by CD14<sup>+</sup> MicroBeads UltraPure human kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's protocol. A total concentration of 1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL was seeded in the wells at a volume of 270 µL per well in cell culture medium containing RPMI, 10% heat-inactivated FCS, 1% 2 mM L-Glutamine, 1% Pen–Strep, and 50 mM 2-mercaptoethanol. The cells were stimulated with 30 µL (final sample concentration 100 µg/mL) of sample and incubated at 37 ◦C for 24 h. Endotoxinfree water was used as the diluent. After incubation, the supernatants were collected and tested for cytokine (IL-6, TNF-α) release by ELISA (Peprotech, London, UK), since these are the prominent cytokines induced by LPS. An LPS standard curve ranging from 1500

to 0.7 pg/mL on the differently coated cytokine ELISA plates was used to determine the quantitative amount of LPS in the samples.
