Synergy neo multi mode microplate reader

Cells were seeded onto 96-well plates at a density of 2 × 10³ cells/well, transfected with 67 mouse DUB siGENOME SMARTpool siRNA library using the Dharmafect I siRNA transfection reagent (Supplementary Data 1), starved overnight, and stimulated with 100 nM insulin for 15 m. Cells were collected and cell lysis was prepared according to the manufacturer’s instructions. All AlphaScreen assays were performed in triplicate with white ½ Areaplate-96 plates (PerkinElmer). For the detection of phosphorylated AKT proteins, anti-P-AKT-coated AlphaScreen SureFire P-AKT1 (pT308) and P-AKT1/2/3 (pS473) acceptor beads (PerkinElmer) were used. Briefly, 16 μl of cell lysate and acceptor beads in buffer (20 μl) were transferred to each well and incubated for 2 h at room temperature. A total of 8 μl of a 1:50 dilution of the streptavidin donor beads was then added to give a final volume of 44 μl, and the mixture was incubated at room temperature for 2 h. All additions and incubations were made in subdued lighting conditions due to the photosensitivity of the beads, and finally, the assay plates were read with a Synergy Neo multi-mode microplate reader (Biotek).

Hep3B and Huh7 Cells were seeded at 1 × 10³ cells/200 ml of medium per well in 96-well plates and treated with or without RNase1 (1 μg/ml) or ALK inhibitors (0.5 μM). After incubation for 48 h, 10 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml) was added to each well, and the cells were incubated at 37 °C for 3 h. Next, 150 ml of DMSO was added to each well to dissolve the water-insoluble purple precipitate. The absorbance in each well was measured at 595 nm with a reference wavelength of 650 nm using a BioTek Synergy Neo Multi-Mode Microplate Reader.

The luciferase activity of B. subtilis reporter strains carrying luxABCDE operon was assayed using a Synergy™ NEO multi-mode microplate reader from BioTek® (Winooski, VT, USA). The reader was controlled by the software Gen5™ (Version 2.06). Luminescence assays were carried out as followed: 10 mL LB medium (w/o antibiotics) were inoculated 1:1000 from overnight cultures (grown with respective antibiotics) and grown to OD₆₀₀ of 0.2–0.3. Then, day cultures were diluted to an OD₆₀₀ of 0.01 and 200 µL were transferred into the wells of a 96-well plate (black wall, clear bottom; Greiner Bio-One, Frickenhausen, Germany). The program was set up for incubation of the plate at 37 °C with agitation (intensity: medium) and the OD₆₀₀ as well as the luminescence was recorded every 5 min for at least 18 h. Luciferase activity (RLU/OD₆₀₀) was defined as the raw luminescence output (relative luminescence units, RLU) normalized to OD₆₀₀ corrected by medium blank at each time point.
If needed, after one hour of incubation, the cells were induced with up to 5 µL of inducer with subtilin supernatant (0.75% (v/v) final concentration) and/or xylose (0.5% (w/v) final concentration). The control samples were treated with the same amount of sterile water. For the “online” reporters, overnight cultures were directly diluted to OD₆₀₀ of 0.01 for luminescence assay without day cultures.