Tristar2 lb 942 microplate reader

Forty-eight hours after transfection, cells were washed with stimulation buffer (Hank’s balanced salt solution, HBSS). For the conformational sensor alone (NY-β2AR), Coel400a, diluted in stimulation buffer, was added (2.5 μM final) for 6 min. Increasing concentrations of ISO, diluted in a stimulation buffer, or 10 μM of different ligands were then added for 5 min. BRET was monitored with a TriSTAR2 LB 942 microplate reader (Berthold Technologies) equipped with a donor filter 485/20 nm and an acceptor filter 530/25 nm. For the multiplexing, the conformational biosensor (NY-β2AR) was co-transfected with the individual transducer biosensors. The response of the NY-β2AR was measured as described above. The transducer biosensors were measured in separate wells. Methozy-e-coelenterazine (0.25 μM) was added for 6 min, followed by incubation with increasing concentrations of ISO, in stimulation buffer, for 5 min (Gs) or 15 min (cAMP production and β-arrestin recruitment). BRET was then monitored with a TriSTAR2 LB 942 microplate reader (Berthold Technologies) equipped with a donor filter of 410/80 nm and an acceptor filter of 515/40 nm. In all cases, BRET ratio was calculated by dividing the acceptor emission over the donor emission.

This assay was used for environmental bacterial isolates which totally inhibit the growth of L. pneumophila on agar plate. A 6-well plate assay was thus designed to physically separate environmental isolates from L. pneumophila Lens in order to determine whether emitted volatiles organic compounds (VOCs) could inhibit or not the growth of L. pneumophila. Each well was filled with 5 mL of BCYE. Then, 10 μL of a suspension of GFP-expressing L. pneumophila Lens (Bigot et al., 2013 (link)) (OD_600nm adjusted to 0.1) were spotted onto both upper sides of the plate. Finally, 40 μL of a suspension of selected isolate (OD_600nm adjusted to 1) were spotted onto the upper center of the plate. Depending of the tested isolate, plates were incubated for 48 h at 22, 30, or 37°C. A second step of incubation was done for 48 h at 37°C. Anti-Legionella activity mediated by VOCs production was quantified by measuring the fluorescence of GFP with a TriStar² LB 942 Microplate Reader (Berthold Technologies, Bad Wildbad, Germany).

For performing MTT viability assays, cells were seeded in a 96-well plate (5 × 10⁴ cells/well for HeLa cells (parental, CK1δ^−/−, or CK1δ^−/− cells stably transfected with wild type or mutant CK1δ), HCT-116, and HT-29 cells or 7.5 × 10⁴ cell/well for SW480 and SW620 cells). After 24 h, old medium was replaced with fresh medium containing either the small molecule of interest or DMSO as control. After 48 h of treatment, 10 μL MTT (5 mg/mL) was added and incubated at 37 °C for 4 h. The medium was then gently removed and 100 μL of acidic isopropanol was added (90% (v/v) isopropanol, 10% (v/v) 1 N HCl). Cell culture plates were then shaken for 30 min at room temperature. Absorbance was measured at 570 nm with TriStar² LB942 Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) and data analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).