Aernoud Fiolet

270 Chapter 11 activation or apoptosis. 18,19 EVs contain bioactive content (proteins, lipids, nucleic acids) from their parent cell and are acknowledged messengers for intercellular communication. 18 We hypothesized that NLRP3 inflammasome stimulation leads to increased NLRP3 protein levels in EVs and that inhibition with colchicine attenuates NLRP3 inflammasome stimulation and reduces circulating EV NLRP3 protein levels and downstream inflammatory markers high sensitivity C-reactive protein (hs-CRP) and IL-6 in patients with chronic coronary disease. MATERIALS AND METHODS Cell culture maintenance, preparation and stimulation Human monocytic cell line of THP-1 cells were obtained from the American Type Culture Collection (ATCC, United States of America) and maintained in Roswell Park Memorial Institute 1640 medium, GlutaMax supplement (Gibco), supplemented with 10% heat-inactivated Fetal Bovine Serum (1 hour at 56°, Biowest) and 1% pen/strep (Gibco) solution). The cell culture mediumwas replaced every two days and cultures were maintained at 37° in a 5% CO2 humidified incubator. NLRP3 inflammasome stimulation in vitro For NLRP3 inflammasome stimulation, THP-1 cells (2 x 106 cells/well) were plated in six-well culture plates in serum-free medium. For differentiating of the THP-1 monocytes into macrophages THP-1 cells were incubated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) for two days. THP-1 cells (non-adherent monocytes or attached macrophages) were incubated with 100 ng/mL of lipopolysaccharide (LPS) (Sigma Aldrich) for three hours. Hereafter, 5 mM of adenosine triphosphate (ATP) (Sigma Aldrich) was added and cells were incubated for another 13 hours for non-adherent monocytes and 3 hours for attached macrophages. For the cells that were treated with colchicine, cells were plated (2 x 106 cells/well) in serum-free medium with either 0.2 µM, 1,0 µM or 10 µMcolchicine 30minutes prior toNLRP3 inflammasome stimulation. Cell viability was examined using trypan blue (Sigma Aldrich) in a trypan blue exclusion assay. The cell culture supernatant was collected and stored at -80°C.