Measuring Odorant Receptor Activation using a Real-Time Cyclic Adenosine Monophosphate Assay

Abstract

Source: Zhang, Y. et al., Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay. J. Vis. Exp.(2017)

This video demonstrates the protocol for measuring odorant receptor activation in transfected cells using a cyclic adenosine monophosphate (cAMP) biosensor and luminescence detection. The method is applied to study receptor-ligand interactions and signaling pathways in olfactory neurons.

Protocol

1. Stimulation and Measuring Odorant Receptor (OR) Activity Using the Real-Time cAMP Assay

1. Observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50-80% per well and return to the incubator.
2. Prepare the stimulation medium by adding 10 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) and 5 mM glucose to Hank's Balanced Salt Solution (HBSS).
3. Thaw the real-time cAMP assay substrate reagent aliquots on ice. Store substrate reagent at -80 °C at 55 µL per tube in polymerase chain reaction (PCR) tubes. Use 1 tube per plate.
4. Prepare 2% equilibration solution by ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMSO	Sigma	D2650
GloSensor cAMP reagent	Promega	E1290
HBSS, w/o calcium chloride and magnesium chloride	GIBCO	14175095
HEPES	Hyclone	SH30237
Muscone	Santa Cruz	sc-200528
Musk tibetene	Sigma-Aldrich	S359165
PBS, w/o calcium or magnesium	Cellgro	21-040-CV
0.2-mL PCR tube	Axygen	PCR-02-C
1.5-mL Eppendorf tube	Eppendorf
15-mL 17 mm x 120 mm conical tube	BD Falcon	352096
8-well and/or 12-well multichannel pipetman	Eppendorf
96-well flat-bottomed white cell culture plate	Greiner	655098
100 mm x 20 mm cell culture dish	BD Falcon	353003
Infinite F200 plate reader	Tecan