This article describes a method for measuring luminescence in recombinant mammalian cells expressing odorant receptors. The technique involves the use of luciferin and a luminometer to assess the activation of these receptors by odorant molecules.
Begin with a multi-well plate containing adherent recombinant mammalian cells in a stimulation solution supplemented with luciferin, a luminescent substrate.
Incubate to allow luciferin entry to the cell.
These cells express membrane-bound odorant receptors coupled with olfactory G-proteins and receptor-transport proteins.
Additionally, the cells express cytoplasmic Glosensor proteins, luminescent proteins sensitive to cyclic AMP.
Add a vaporizable odorant solution in the space between the wells and place the plate within a luminometer instrument pre-equilibrated with the same odorant.
Initiate luminescence measurement.
Odorant molecules bind to the cells' odorant receptors, activating the coupled G-protein and dissociating the G-protein’s α subunit, or Gα.
The dissociated Gα subunit binds to the membrane-embedded adenylate cyclase enzyme, activating it. The activated enzyme converts the ATPs to cyclic AMPs.
These cyclic AMPs bind with glosensor proteins, altering their conformation and enabling the luciferin binding.
This interaction produces luminescence, confirming the activation of odorant receptors.
Add 75 microliters of the cAMP reagent solution. Remove the stimulation solution from the wells and add 25 microliters of diluted cAMP reagent solution to each well. Incubate the 96 well plate at room temperature in a dark and odor free environment for two hours.
First, dilute the odorant acetophenone to 1% in 10 milliliters of mineral oil and cap the tube. Before the end of the cAMP reagent incubation time, add 25 microliters of the odorant solution to each well in a new 96 well plate. Then place this odorant plate in the luminometer chamber for five minutes to equilibrate the chamber with volatile odorant molecules. Set the luminometer to record the luminescence with zero seconds of delay during 20 cycles of 90-second plate measurement, with 0.7 seconds of interval between cycles.
Right before reading the plate, remove the odorant plate from the chamber. In the 96 well plate containing the transfected cells, add 25 microliters of odorant in the space between the wells, and quickly put the plate back into the chamber. Start the luminescence measurement of all wells for 20 cycles within 30 minutes.
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