Synergy 2 plate reader

The expression of lux-based reporters from cells grown in liquid culture was measured as counts per second (cps) of light production in a Synergy 2 plate reader (Biotek). The pKD-related plasmids were electroporated into P. aeruginosa recipient strains. Overnight cultures of bacteria were subcultured at a 2% dilution with fresh LB medium with 15 mM KNO₃ added, and then the cells were grown under aerobic and anaerobic conditions up to an OD₆₀₀ of ≈1.0. The cultures were added to a black 96-well plate with a transparent bottom. The bacterial luminescence value and the OD₆₀₀ value were measured in a scanning mode in a Synergy 2 plate reader (Bio Tek), and the promoter activity was measured at a ratio of cps to OD₆₀₀ (relative luminescence value).

CyQUANT assay is based on the measurement of cellular DNA content via fluorescent dye binding to cellular DNA. CyQUANT assay was carried out as per manufacturer’s instructions (Invitrogen). Briefly, ONS cells in black-walled 96 or 384 well culture plates (Nunc) were washed twice with HBSS buffer (Invitrogen) and either 50 μL or 12.5 μL of reaction mixture containing (1X CyQUANT dye reagent and 1X dye delivery reagent) was added in each well and incubated at 37°C for 90 min, then the fluorescence intensity of each sample was measured using a Synergy II plate reader (BioTek) with excitation at ~485 nm & emission detection at ~530 nm.

Cells were plated at 40% confluency in 96-well, flat bottom plates. After being allowed to attach overnight, cells were treated with vehicle JNK-IN-8, lapatinib, or JNK-IN-8 and lapatinib combination for various timepoints. After treatment, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (#475989 Calbiochem, San Diego, CA) was added to a final concentration of 0.5mg/ml from 5mg/ml stock in PBS. Cellular metabolism reduces this tetrazole to insoluble formazan crystals. After four hours of incubation, the media + MTT was gently removed. Formazan crystals were dissolved using DMSO and absorbance at 590nm was read using the Synergy II Plate Reader (BioTek Winooski, VT). For viability assays using MTS, the Promega (Madison, WI) CellTiter 96® AQ_ueous One Solution was used according to manufacturer's instructions. Absorbance values were read at 490nm using the Victor³V Model 1420 plate reader from PerkinElmer (Waltham, MA).

HEK293T cells were seeded in 12-well plates and transfected with AVPR1A-hRluc alone or together with plasmids encoding EYFP or ACKR3-EYFP using the Lipofectamine 3000 transfection reagent (ThermoScientific). For BRET titration assays, AVPR1A-hRluc at the fixed amount of 50 ng was co-transfected with increasing amounts of EYFP or ACKR3-EYFP. For BRET assays at a constant acceptor : donor ratio, increasing amounts of AVPR1A-hRluc and ACKR3-EYFP were co-transfected at a ratio of 1 : 10. In all assays, empty vector pcDNA3 was added to maintain the total cDNA amount for each transfection reaction constant. After an overnight incubation, cells were seeded in poly-l-lysine coated 96-well white plates and incubated again overnight. Cells were then washed with PBS and fluorescence was measured in a Biotek Synergy II plate reader (λ_excitation 485 nm, λ_emission 528 nm). For BRET measurements, coelenterazine H (Nanolight Technology) at 5 µM in PBS was added to the cells. After 10 min incubation at room temperature, luminescence was measured at 460 ± 40 and 528 ± 20 nm. The BRET signal is calculated as the ratio of RLU measured at 528 ± 20 nm over RLU at 460 ± 40 nm. The net BRET is calculated by subtracting the BRET signal detected when the AVPR1A-hRLuc was transfected alone. For titration experiments, net BRET ratios are expressed as a function of EYFP/total luminescence.

The PRESTO-Tango (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation) assay was performed as recently described [27 (link)]. The Tango plasmids were a gift from Dr Bryan Roth (all from Addgene). HTLA cells (2.5 × 10⁵ per well) were seeded in a six-well plate and transfected with 1.5 µg of the Tango plasmids using Lipofectamine 3000 (ThermoScientific). The following day, transfected HTLA cells (1 × 10⁵ cells per well) were plated onto poly-l-lysine precoated 96-well microplates and allowed to attach to the plate surface for at least 4 h prior to treatment. Proteins used for treatment were prepared in twice the final concentration in culture media, added at a 1 : 1 vol/vol ratio and incubated overnight at 37°C, 5% CO₂ in a humidified environment. The following morning, media were removed from cell culture plates and replaced with a 100 µl 1 : 5 mixture of Bright-Glo (Promega) and 1× HBSS, 20 mM HEPES solution. Plates were then incubated at room temperature before measuring luminescence on a Biotek Synergy II plate reader.