*5.8. Incubation of THP-1 Macrophages with BaV and Kn-Ba*

THP-1 differentiated in 24-well plates, according to what is described in 5.4, were treated with BaV (0.5 μg, 1 μg, 10 μg, and 15 μg/well) or with Kn-Ba (0.5 μg and 1 μg/well) diluted in 500 μL/well of supplemented RPMI medium without FBS and incubated at 37 ◦C in an atmosphere containing 5% CO2 during 24 h, 48 h, and 72 h [66]. The supernatants were collected, centrifuged (405× *g*) for 5 min at 4 ºC for removal of cell debris, aliquoted, and stored at −80 ◦C.

#### *5.9. Release of Lactate Dehydrogenase (LDH)*

The cytotoxic effects after BaV and Kn-Ba treatments were evaluated by the quantification of lactate dehydrogenase (LDH) enzyme in cell-free supernatants using the kit CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) [67], according to the manufacturer's instructions. Cell-free supernatants of THP-1 macrophages treated during 45 min with lysis buffer were used as a positive control of LDH release, and cells treated only with culture medium were used as LDH release background.
