Arvo microplate reader

Cell proliferation was estimated by a DNA assay. In brief, OVCAR3, RMG1, and SKOV3 cells plated onto 96-well plates were transfected with siRNAs and then incubated for 4 days. A DNA assay was performed using the fluorochrome Hoechst 33258 to quantify cellular DNA content in 96-well tissue cultures plates [57 (link)]. Five replicate wells (n = 5) were used for the cell proliferation assay. Fluorescence intensity was measured using an ARVO microplate reader (PerkinElmer, Foster City, CA, USA).

Ozoralizumab was generated at Wyeth Research in the manner previously described (37 (link)). Adalimumab was purchased from Eisai Co., Ltd (Japan). Recombinant human TNFα alpha was purchased from R&D Systems (United States). All samples were diluted with PBS (Thermo Fisher Scientific, United States) or saline (Otsuka, Japan). A 20.3 μM solution of ozoralizumab or Adalimumab was mixed with 2.3 μM, 6.6 or 6.8 μM, 20.3 μM, or 60.8 μM solution of TNFα trimer and incubated overnight at 4°C. Mixtures of the respective antibodies and TNF were added to mouse neutrophils to a final antibody concentration of 33-34 nM. Reactive Oxygen Species (ROS) were measured by using L-012 sodium salt (8-Amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d] pyridazine sodium salt) (38 (link)). Neutrophils (5 x 105 cells/well) in CL medium containing 50 μM L-012 (FUJIFILM Wako Pure Chemical, Japan) were transferred to white microplates (Greiner Bio-One, Austria), and ROS-dependent chemiluminescence was measured with an ARVO microplate reader (PerkinElmer, United States). ROS released by the neutrophils were monitored for 1 min at 37°C.