Materials and Reagents

White, Tissue Culture-Treated (TC) 96-well plates (Corning, catalog number: 3917 )
Non-binding V bottom plates (for serial dilutions; for example, Costar, catalog number: 3896 )
Tissue Culture-Treated (TC) flasks (growth area 150 or 75 cm²; Falcon, catalog number: 355001 or 353107 )
HEK293 cells
0.05% Trypsin/EDTA (Life Technologies, catalog number: 25300 )
Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, catalog number: 11995 )
Fetal Bovine Serum (FBS) (HyClone, catalog number: SH30070.03 )
100x Penicillin-streptomycin (Sigma, catalog number: P4333 )
Opti-MEM without Phenol Red (Life Technologies, catalog number: 11058 )
FuGENE HD (Promega, catalog number: E2311) or ViaFect (Promega, catalog number: E4981 )
Note: Other transfection reagents can also be used.
DMSO (Sigma, catalog number: D2650-5X5ML )
Tracer dilution buffer (Promega, catalog number: N2191 )
Nano-Glo HiBiT Extracelular Detection System (Promega, catalog number: N2420 or N2421 )
Transfection Carrier DNA (Promega, catalog number: E4881 )
Test compound of interest
DNA construct encoding a HiBiT- tagged GPCR fusion
Note: HiBiT-tagged GPCR constructs can be obtained from Promega Custom Assays Systems or generated via cloning into pFN39K secHiBiT CMV-neo Flexi Vector (Promega, catalog number: N2411 ) or pBiT3.1-secN [CMV/HiBiT/Blast] vector (Promega, catalog number: N2381).
Fluorescent Tracer
Note: Several NanoBRET GPCR Tracers can be obtained from Promega Custom Assays Systems. In addition a fluorescent GPCR Tracer can be generated according to the published protocol (Robers et al., 2019) using building blocks that can be obtained from Promega Custom Assays Systems.
Miscellaneous tissue culture reagents
DMEM Cell Culture Medium (see Recipes)
Opti-MEM Assay Medium (see Recipes)

Equipment

BRET-compatible microplate luminometer equipped with 450 nm (bandpass) and 600 nm (longpass) filters (e.g., Promega GloMax Discover, PerkinElmer EnVision, or BMG Clariostar)
Orbital plate shaker
Miscellaneous tissue culture equipment

Software

GraphPad Prism (https://www.graphpad.com/scientific-software/prism)

Procedure

Transient transfection of HEK293 cells with HiBiT-tagged GPCR constructs
Note: The transfection conditions described below are recommended for adequate translocation and cell surface expression of HiBiT-tagged GPCRs. These include:
1. Dilution of DNA encoding the HiBiT-tagged GPCR into a promoterless Carrier DNA. This maintains the total amount of transfected DNA but allows for lower expression levels across the population of transfected cells. A 100-fold dilution is generally recommended, however in some cases this might require further optimization.
2. DNA: Transfection reagent complexes should account for 10% (vol/vol) of transfected cell culture.
1. Cultivate HEK293 cells in Culture Medium (Recipe 1) in a TC-75 or TC-150 flask prior to assay.
  Note: Cell density at time of harvest can affect transfectability. For optimal transfectability use freshly passaged HEK293 cells (ideally within 1-2 days) at 80-95% confluency.
2. Aspirate medium from flask, add 5 ml or 8 ml trypsin to TC-75 or TC-150 flask, respectively and allow cells to dissociate from the flask.
3. Neutralize trypsin using 20 ml or 25 ml Culture Medium (for TC-75 or TC-150, respectively), and pellet cells via centrifugation at 200 x g for 5 min.
4. Aspirate medium and resuspend cells into a single cell suspension using Opti-MEM Assay Medium (Recipe 2).
5. Adjust the cell density to 2.2 x 10⁵cells/ml in Opti-MEM Assay Medium in a sterile, conical tube.
6. Prepare transfection reagent: DNA complexes.
  Below is an example for transfection of 10 ml culture:
  1. Prepare a 10 µg DNA solution in Opti-MEM without serum. This solution should contain the following ratios of carrier DNA and DNA encoding the HiBiT-tagged GPCR construct:
    1. 9.9 µg/ml of Transfection Carrier DNA.
    2. 0.1 µg/ml of DNA encoding the HiBiT-tagged GPCR (100-fold dilution).
    3. 0.5 ml of Opti-MEM without serum.
  1. Mix thoroughly.
  2. Combine 30 µl transfection reagent (i.e., FuGENE HD or ViaFect) with 0.5 ml Opti-MEM without serum and mix by pipetting.
    Note: If using another transfection reagent, follow manufacturer recommendation.
  3. Combine the DNA and transfection reagent solutions and mix by pipetting 10-15 times.
  4. Incubate at room temperature for 15 min to allow transfection reagent, DNA complexes to form.
7. Combine one part (e.g., 1 ml) of transfection reagent: DNA complexes with 9 parts (e.g., 9 ml) of HEK293 cells in suspension at 2.2 x 10⁵cells/ml. Mix gently by inversion 5 times and plate in a white TC-treated 96-well plate at 95 µl/well.
  Note: Larger or smaller bulk transfections should be scaled accordingly, using this ratio.
8. Incubate for at least 20 h at 37 °C + 5% CO₂.

NanoBRET GPCR-ligand engagement protocols
1. Treat cells with a serial dilution of a fluorescent Tracer.
  1. Prepare a 20x serially diluted Tracer.
    1. First prepare a 100x serially diluted Tracer in DMSO. Prepare 3-fold serial dilutions starting at 300 µM, which corresponds to a final 1x concentration of 3 µM. In some cases, a higher or lower Tracer concentration may provide a better assay window.
      Note: Make sure to include within the serial dilution a no Tracer control.
    2. Dilute the Tracer Dilution Buffer (TDB) 3-fold in Opti-MEM without phenol red to generate a 0.3x TDB.
    3. Dilute the 100x serially diluted Tracer 5-fold in 0.3x TDB to generate a 20x serially diluted Tracer.
  2. Add 5 µl of 20x serially diluted Tracer per well of transfected cells (Figure 2 portrays a 96-well plate set-up).
  3. Mix briefly on an orbital plate shaker.
2. To determine specific binding treat control cells with excess unmodified compound.
  1. Prepare a 20x unmodified compound solution.
    1. Prepare a 10% (vol/vol) DMSO solution in Opti-MEM without phenol red.
    2. Prepare a 600 µM unmodified compound solution in 10% DMSO, which corresponds to a final 1x concentration of 30 µM.
  2. Add 5 µl of the 20x unmodified compound solution to the control wells (Figure 2 portrays a 96-well plate set-up).
  3. Mix briefly on an orbital plate shaker.
  4. Incubate plate at room temperature (to minimize receptor internalization) for 90 min protected from light, then proceed to NanoBRET detection section below.
    Note: Agonist binding promotes receptor internalization to different extents, which can be detected through dose dependent decrease in donor signal (e.g., signal at 450 nm). Still, binding experiments at room temperature (22-25 °C) decease the rate of internalization and the ratiometric nature of BRET further minimizes the influence of internalization on binding analyses. Accordingly, binding analyses at 37 °C may require a shorter incubation time.
  Figure 2. A 96-well plate set-up for saturation binding assay. Cells treated with serial dilution of a fluorescent Tracer in the absence and presence (control wells) of excess unmodified compound.
3. Detect NanoBRET
  1. Immediately prior to BRET measurements prepare 2x HiBiT detection reagent in Opti-MEM without phenol red. This reagent consists of 100-fold dilution of LgBiT and 50-fold dilution of Nano-Glo HiBiT Extracellular Substrate. Mix thoroughly by pipetting.
  2. Add 100 µl detection reagent/well.
  3. Mix on an orbital plate shaker for 15 min.
  4. Measure donor emission (e.g., 450 nm) and acceptor emission (e.g., 610 nm or 630 nm) using a NanoBRET-compatible luminometer.
4. Determine BRET ratio and process data.