The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

1. Production and evaluation of the HiBiT-RBD bioreporter

1. Producing a sufficient quantity of HiBiT-RBD bioreporter
   1. Prepare for cell culture
      1. Prepare complete Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Then, warm the media in a 37 °C water bath.
      2. Turn the biological safety cabinet (BSC) on and use 70% v/v ethanol for sterilizing the cabinet surface.
   2. Culture the cell line.
      1. Take out the cell line from -80 °C or liquid nitrogen and thaw it in a 37 °C water bath.
         NOTE: An appropriate cell line is easy to maintain in culture, has high transfection efficiency, and is suitable for exogenous protein production. Human embryonic kidney, HEK293 cells were used for this protocol.
      2. Mix the thawed cells with at least 10 mL of complete medium, pipette the cell suspension to a 10 cm Petri dish, and swirl the plate to distribute cells in the dish uniformly. Place the dish in a cell culture incubator at 37 °C, 5% CO₂, and 85-95% humidity.
      3. Observe the cells under the microscope until the confluency level reaches 80-90%. At high confluency, remove the medium, wash the cells with warm phosphate-buffered saline (PBS), and add 1 mL of 0.25% trypsin-ethylenediamine tetra acetic acid to detach the cells from the surface.
         NOTE: The HEK293 cells are fairly easily detached. Hence, the washing step should be done very gently to prevent accidental detachment and loss of cells.
      4. After approximately 5 min, look at the cells under a microscope. If all cells are floating, add at least 4 mL of medium, and transfer the cell suspension into a new sterile tube. Count the cells using a hemocytometer and add 1 × 10⁶ cells into each well of a 6-well plate for transfection.
         NOTE: After 24 h, cells should be at 80% or more confluent.
   3. Transfection of the HiBiT-RBD plasmid
      1. Use 1 µg of the HiBiT-RBD expression plasmid with a suitable transfection reagent. Incubate for 10-15 min at room temperature, and then add the total volume to each well of the plate, drop-wise.
         NOTE: Follow the manufacturer's transfection protocol. In this case, a mixture (a specified amount) of the transfection reagent with DMEM was added to the diluted plasmid (1 µg) in DMEM (see the Table of Materials). Use a marker containing (e.g., green fluorescent protein [GFP]) plasmid as a control to monitor the transfection efficiency.
      2. On the next day, replace the medium containing the transfection mixture with complete medium. Observe the transfection control well 48 h after transfection.
         NOTE: If the transfection was positive and efficient (more than 80% GFP-positive), the cells should be ready for harvesting the HiBiT-RBD bioreporter. The construct also contains His-tag, which can be used to obtain purified protein in place of the total supernatant.
      3. Collect the supernatant in 1.5 mL microtubes. Add 500 µL of 1x passive lysis buffer (PLB) to the cells; incubate and shake the plate for 15 min at room temperature for cell lysis.
         NOTE: The supernatant and the lysate solution can be preserved at -20 °C with minor loss of integrity for at least 6 months. The reporter is stable at a wide range of pH (4-12) and temperature (4-42 °C).
2. Evaluation of the luminescent signal from the bioreporter by luciferase assay
   1. Preparing the reaction components
      1. Use the supernatant as the source of the bioreporter.
         NOTE: Both supernatant and lysate contain the HiBiT-RBD bioreporter and can be used for the assay. However, the supernatant is recommended as the source.
      2. Dilute the LgBiT and substrate to 1x before use (stock concentration is 100x).
         NOTE: See the Table of Materials for details about the LgBiT.