Ensight perkin elmer multimode plate reader

The DELFIA assay was performed to determine cell proliferation according to the manufacturer’s protocol from PerkinElmer (Milan, Italy). The assay is a time-resolved fluoroimmunoassay based on the incorporation of BrdU into newly synthesized DNA strands of proliferating cells cultured in microliter plates. Incorporated BrdU is detected using a europium labeled monoclonal antibody and the fluorescence measured is proportional to the DNA synthesis in the cell population of each well. A375, A549, and MRMT-1 cells were cultured over night at 1000 cells/well in a 96-well plate (at a final volume of 100 μl per well), agonists and antagonists were added and the cells were incubated for 30′ before addition of the BrdU-Labeling solution 10 μl/well. The cells were then cultured for 24, 48, or 72 h. At the end of the incubation period, cells were fixed (fix solution 100 μl/well), added with 100 μl/well of Anti-BrdU-Eu (0.5 μg/ml) and incubated for 120 min at room temperature. After four washes, 200 μl of DELFIA Inducer were added at room temperature for 15 min and the Eu-fluorescence was detected through an Ensight Perkin Elmer-multimode plate reader (Perkin Elmer, Milan, Italy). Two kinds of controls were performed: the blank where no cells were added to the well but only culture medium and the background where no BrdU was added to the wells.

AlphaScreen SureFire phospho(p)ERK1/2(Thr202/Tyr204), pJNK1/3(pThr183/Tyr185), and p-AKT1/2/3 (pThr308) assay kits (Perkin Elmer, Milan, Italy) were utilized. Upon kinase phosphorylation and excitation at 680 nm, fluorescent signals at 615 nm are emitted. Cells were seeded in 100 μl culture medium into 96-well plates (30,000/well), and incubated at 37°C for 24 h. Cells were pretreated with various inhibitors and TP455 for 30 min. Then, receptors were maximally stimulated using 100 nM CGS 21680 and incubated for 5 min (ERK1/2, JNK1/2-MAPK) or 30 min (AKT) at 37°C. After agonist removal, lysis buffer was added, then donor and acceptor beads linked to specific anti-p-kinase- and anti-kinase-antibodies were dispensed, according to manufacturer instructions. Finally, fluorescent signals were detected through an Ensight Perkin Elmer-multimode plate reader (Perkin Elmer, Milan, Italy). Data were normalized to fold activation above basal p-kinase levels (=100). For inhibitor graphs, raw data were transformed into percentages relative to controls (basal level = 100%) in order to merge data from several experiments.