1. Transient transfection of the expression plasmids into the cells

1.  Harvest the adherent HEK293T cell culture by trypsinization and resuspend the cells in a dedicated complete growth medium. Plate the cells (2 x 10⁴/100 µL/well) onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls including replicates.
   NOTE: Attempt to use only the inner 60 wells of the plate to minimize thermal shifts and avoid overnight evaporation. Using poly-D-lysine-coated plates is highly recommended such as the ones indicated in the Table of Materials, otherwise, cells may detach during the subsequent washing steps.
2. Culture the cells overnight in standard conditions (37 °C, 5% CO₂).
3. On the next day transfect the cells with the desired combinations of expression plasmids.
   1. Dilute expression plasmids in a serum-free medium (see Table of Materials) to 6.25 ng/µL for each construct.
   2. Add the lipid-based transfection reagent at an appropriate lipid-to-DNA ratio and incubate according to the manufacturer’s instructions.
   3. Add 8 µL of lipid: DNA mixture to designated wells. Mix the content of the plate by gentle rotation. This results in the transfection of both expression constructs at 50 ng/well.
4. Culture the cells for 20-24 h in standard conditions (37 °C, 5% CO₂).
   NOTE: Culturing the cells for a longer time may result in higher levels of fusion protein expression, which may promote a non-specific association between the fragments.

2. Medium exchange

1. On the next day replace the conditioned medium with 100 µL of a serum-free medium in each well. Make sure that the cells have not detached upon medium exchange.
   NOTE: This step should be done 2-3 h before the addition of the furimazine working solution. Serum withdrawal minimizes background caused by auto-luminescence of furimazine.

3. Preparation of furimazine working solution

1. Just before the measurement, mix 1 volume of furimazine with 19 volumes of a dilution buffer (a 20-fold dilution).
   NOTE: The total volume of the furimazine working solution to be prepared depends on the number of individual wells to be analyzed (the furimazine working solution is added to the cell culture medium in a 1:5 ratio, therefore, to each well previously filled with 100 µL of a serum-free medium 25 µL of the furimazine working solution should be added).
2. Add the furimazine working solution to designated wells (25 µL/well). Gently mix the plate by hand or using an orbital shaker (e.g., 15 s at 300-500 rpm).

4. Measuring luminescence

1. Insert the plate into a luminescence microplate reader.
   1. For experiments that are to be performed at 37 °C equilibrate the plate for 10-15 min at the indicated temperature.
2. Select the wells to be analyzed.
3. Read luminescence with an integration time of 0.3 s. Continue to monitor luminescence for up to 2 h when required.