This article describes a method for measuring the activation of olfactory receptors in transfected mammalian cells using a cAMP biosensor. The process involves preparing the cells, adding odorants, and measuring luminescence to assess receptor activity.
Take a multiwell plate containing transfected mammalian cells.
These cells express the olfactory neuron's odorant receptor (OR), a G-protein-coupled receptor, along with a cAMP biosensor containing a cAMP binding domain fused to a mutant luciferase.
Remove the medium and wash with buffer to remove the residual medium.
Add a buffer containing a cAMP biosensor substrate. Incubate to facilitate substrate uptake into the cells.
Using a chemiluminescence plate reader, measure the basal luminescence of the cells.
Add increasing concentrations of the test odorant to the wells, leaving one well untreated as a control.
The odorant binds to the OR and triggers a signaling cascade that produces cAMP.
This cAMP binds to the biosensor's binding domain, activating luciferase, which interacts with the substrate to emit light.
Measure the luminescence in real time. An increase in luminescence with higher odorant concentrations indicates OR activation by the odorant.
Before stimulation, observe the transfected cells under a phase-contrast microscope to ensure a proper confluence of 50% to 80% per well before returning to the incubator. Then, prepare stimulation medium by adding 10 millimolar HEPES and 5 millimolar glucose to Hank's Balanced Salt Solution. Thaw 55-microliter aliquots of real-time cyclic AMP assay substrate reagent aliquots on ice.
Prepare 2% equilibration solution by mixing 55 microliters of the substrate reagent and 2,750 microliters of stimulation medium. After spreading out a thick layer of paper towels on the bench, gently and repeatedly tap the plate upside down on the paper towels so that the transfection medium is absorbed by the paper towels. Wash the cells by pipetting 50 microliters of stimulation medium to each well.
Gently and repeatedly tap out the stimulation medium from the 96-well plate. Pipette 25 microliters of 2% equilibration solution into each well and incubate at room temperature in the dark for 2 hours. Prior to the end of the incubation time, dilute thawed odorant stock solutions to working concentration in stimulation medium.
Before odorant addition, use a chemiluminescence plate reader to measure the basal luminescence level of the plate. Quickly remove the plate from the plate reader and add 25 microliters of the odorant dilutions to each well. Then, immediately start continuous luminescence measurement of all wells for 20 cycles within 30 minutes.
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