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 mixing 55 µL of the substrate reagent and 2750 µL HBSS/HEPES/glucose solution.
5. Spread a thick layer of paper towels on the bench. Gently and repeatedly tap the plate upside-down on the paper towel so that the transfection medium is completely absorbed by it.
6. Wash the cells by pipetting 50 µL of HBSS/HEPES/glucose solution to each well.
7. Gently and repeatedly tap out the HBSS/HEPES/glucose solution from the 96-well plate.
8. Pipette 25 µL of 2% equilibration solution to each well and incubate at room temperature in the dark for 2 h.
9. Prepare in advance 1 M odorant stock solutions in dimethyl sulfoxide (DMSO) and store at -20 °C until used.
10. Prior to the end of the incubation time, dilute the odorant stock solutions to working concentrations in the HBSS/HEPES/glucose stimulation medium.
    NOTE: The concentrations of the odorant dilutions prepared in this step should be doubled to yield the correct final concentrations in each well.
11. Using a chemiluminescence plate reader and before odorant addition, measure the basal luminescence level of the plate for 2 consecutive times at a rate of 1000 ms per well.
12. Quickly remove the plate from the plate reader, add 25 µL odorant dilutions to each well, and immediately start continuous luminescence measurement of all wells for 20 cycles within 30 min.
    NOTE: Pipette carefully to avoid contaminating neighboring wells when using different odorants and/or different concentrations of the same odorants in the same plate.

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