BRET and CODA-RET assays

HEK293 cells (2 × 10⁵ per well of a 6-well plates) were transfected with 2 ng plasmid DNA coding for the BRET donor (CD147-Luc, CD147ΔD2-Luc) and increasing amounts of BRET acceptor plasmids (β₂-YFP, CCR5-YFP; 0–400 ng per well). Twenty-four hours after transfection, cells were resuspended in Hank's balanced salt solution and distributed in 96-well plates (Perkin-Elmer plates; 10⁵ cells per well). After addition of the luciferase substrate, coelenterazine h (5 μM final concentration), luminescence at 485 nm and fluorescence at 530 nm were measured simultaneously in a Mithras LB940 Plate Reader. The BRET ratio was calculated as: ((emission at 530 nm/emission at 485 nm)−(background at 530 nm/background at 485 nm)), where background corresponds to signals in cells expressing the Rluc fusion protein alone under the same experimental conditions. For better readability, results were expressed in milli-BRET units (mBRET), 1 mBRET corresponding to the BRET ratio multiplied by 1000. BRET ratios were plotted as a function of ([YFP-YFP₀])/(Rluc), where YFP is the fluorescence signal at 530 nm after excitation at 485 nm, and Rluc the signal at 485 nm after addition of coelenterazine h. YFP₀ and Rluc₀ correspond to the same values in cells expressing the Rluc fusion protein alone. For the β₂AR stimulation assays, after reading of the basal energy transfer, 10 μl of vehicle, isoproterenol (agonist) or propranolol (antagonist) at a final concentration of 10 μM was added per well and the energy transfer was measured during 20 min. To address the effect of bacterial adhesion, after reading of the basal energy transfer, bacterial suspension or purified meningococcal pilins MBP-PilE or MBP-PilV (10 μg ml⁻¹) were added and the energy transfer was measured during 20 min.

To perform the CODA-RET assays, HEK293 cells (1 × 10⁵ per well of a 12-well plates) were transfected with plasmids DNA coding for the BRET donors (900 ng CD147-L1 and 100 ng β₂AR-L2) and increasing amounts of BRET acceptor plasmids (Actn4-YFP or YFP alone; 0–400 ng per well). Twenty-four hours after transfection, cells were resuspended in Hank's balanced salt solution and distributed in 96-well plates (Perkin-Elmer plates). After addition of the luciferase substrate, coelenterazine h (5 μM final concentration), luminescence at 485 nm and fluorescence at 530 nm were measured simultaneously in a Mithras LB940 Plate Reader and BRET ratio was calculated and plotted as above.