Nanoglo hibit detection system

The number of receptors expressed on the cell membrane as well as total cell expression were determined using the NanoGlo^® HiBiT detection system (Promega, Mannheim, Germany). Here, HEK293 cells express the HiBiT-tagged receptor in low amounts and measurement was performed according to the manufacturer’s protocol (rapid measurements protocol). In short, 48 h after transfection, media was changed to 50 µL Opti-MEM without phenol red per well and injected with 50 µL of either HiBiT Extracellular substrate (Promega, Mannheim, Germany) for determination of cell surface expression or HiBiT Lytic substrate (Promega, Mannheim, Germany) for total expression using a plate reader (Mithras LB 940, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Afterwards, the plate was shaken orbitally for 3 min at 300 cycles per minute, incubated for 10 min at room temperature and then luminescence was measured. Cells transfected with empty pcDNA3 were used as background control and were subtracted from the sample emissions.

The Nano-Glo^® HiBiT detection system (Promega, Mannheim, Germany) was used to quantify the cell surface and the total expression of MC4R [12 (link)]. Measurements were performed according to the manufacturer’s protocol. Two days after transfection, the medium was changed to 50 µL/well Opti-MEM without phenol red, and 50 µL of either HiBiT extracellular substrate (Promega, Mannheim, Germany) or HiBiT lytic substrate (Promega, Mannheim, Germany) was added. After orbital shaking for 3 min at 300 cycles/min and incubation at room temperature for 10 min, luminescence was measured using a Berthold Microplate Reader (Mithras LB 940, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). HEK-293 cells transfected with the empty vector pcDNA3 served as background control.

The amount of receptors expressed on the cell membrane as well as total cell expression was determined using the NanoGlo® HiBiT detection system (Promega). Assay was performed according to manufacturer’s protocol (rapid measurements) and has been described elsewhere.^{47 (link)} In short, 48 h after transfection, media were changed into Opti-MEM reduced serum media (Thermo Fisher Scientific, Gibco) without phenol red (50 µL/well) to remove background noise. Determination of cell surface expression was performed by injection of 50 µL of HiBiT extracellular substrate in the appropriate buffer supplemented with LgBiT. Total expression determination was carried out similarly, with 50 µL/well of HiBiT Lytic substrate combined with LgBiT in the appropriate buffer containing detergents to lyse the cells (Promega). After orbital shaking for 3 min at 300 cycles per minute, plates were incubated for 10 min at room temperature. Luminescence was measured using a plate reader (Mithras LB 940, Berthold Technologies). As background control, cells transfected with empty vector pcDNA3 were used and values were subtracted from the sample emissions.