Nano glo live cell reagent

HEK293T cells were seeded in 96-well plates in 10%FBS DMEM and transfected 24 h later with 2.5 ng pLgBiT-MR (LgBiT at N-terminus) and 2.5 ng pGR-SmBiT (SmBiT at C-terminus) for the GR-MR heterodimerization assay; or 1.5 ng pLgBiT-GR (LgBiT at N-terminus) and 1.5 ng pGR-SmBiT (SmBiT at C-terminus) for the GR-GR homodimerization assay; or 1 ng pLgBiT-MR (LgBiT at N-terminus) and 1 ng pMR-SmBiT (SmBiT at C-terminus) for the MR–MR homodimerization assay, using calcium phosphate precipitation. 24 h later, the Nano-Glo® Live Cell reagent was reconstituted (Promega) and 25 μL was added to the transfected cells, after which the baseline luminescence was measured for 15 min (continuous-mode, 1-min intervals) using an Envision (PerkinElmer) spectrophotometer. Subsequently, ligands were added (see Fig. legends) and the luminescence was measured in a time window of 60 min (continuous-mode, 1-min intervals). Luminescence counts were normalized to baseline and set as a fold-difference versus the solvent condition (here: DMSO). The area under the curve method was used to statistically compare Dex and Dex-Spi conditions.

BHK21 cells in growth medium were seeded in white 96-well plates (Costar 3917) at 37 °C and 5% CO₂. The cells were co-transfected with pcDNA3.1-C-UL47-LgBiT and pcDNA3.1-N-STAT1-SmBiT using Lipofectamine 3000 according to the instructions; FKBP-SmBiT and FRB-LgBiT were used as positive controls, whereas Halotag-SmBiT coexpressed with pcDNA3.1-C-UL47-LgBiT, pcDNA3.1-C-UL47-LgBiT coexpressed with FKBP-SmBiT and pcDNA3.1-N-STAT1-SmBiT coexpressed with FRB-LgBiT were used as negative controls. The medium was replaced with serum-free media, and luciferase was detected after 20 h posttransfection. Luminescence was measured after addition of 25 μl Nano-Glo Live cell reagent (Promega, N2012) prepared according to the manufacturer’s protocol. All reactions were performed in triplicate and in at least three independent experiments.

To assess whether the HiBiT-FLAG fragment, HiBiT-FLAG-BepD_Bhe, and HiBiT-FLAG-BepD_Bta can complement LgBiT to a functional luciferase, we used the Nano-Glo HiBiT lytic detection system (Promega, Cat. N3030). Bacteria were cultured as previously indicated. Around 5 × 10⁸ bacteria were resuspended in 100 μl PBS and supplemented with 100 μl LSC buffer containing the substrate (1:50 v/v) and the LgBiT protein (1:100 v/v). The luminescent signal was measured using the Synergy H4 plate reader.
RAW LgBiT macrophages were infected as described previously. Effector translocation into macrophages was quantified by measuring the luminescent signal using the Synergy H4 plate reader. The Nano-Glo live cell reagent (Promega, Cat. N2011) was prepared as advised by manufacturer. After 24 h of infection, supernatant was aspirated and cells were gently washed in pre-warmed PBS. A final volume of 100 μl PBS per well was supplemented with 25 μl of the Nano-Glo live cell assay buffer containing the substrate for luminescence measurement in the Synergy H4 plate reader. The following settings were used: temperature 37°C, shaking sequence 30 s at 300–500 rpm, delay 10 min, autoscale, and integration time 5 s. At least, three independent experiments (n = 3) were performed in technical triplicates.

To detect ligand-induced interaction between GPR132 and β-arrestin, a NanoBit^® β-arrestin recruitment assay was performed [23 (link)]. HEK293T cells were co-transfected GPR132-LgBit and β-arrestin2-SmBit vectors at 1:1 and seeded into white 96-well plates (with transparent bottom) at a density of 2 × 10⁴ cells per well. Twenty-four hours later, cells were loaded with Nano-Glo Live Cell Reagent (Promega, Madison, WI, USA) in a CO₂-independent medium at 37 °C. Then, compound stock solutions were dispensed to 96-well plates, and luminescence signals were monitored continuously for 1 h or more at 37 °C with a Cytation^TM 5 Imagine Multi-Mode Reader (BioTek, Winooski, VT).