The overall goal of this real-time high-throughput cyclic AMP assay is to screen odorants against odorant receptors and vice versa. Since mice are useful for investigating odorant receptor functioning in that it allows for larger scale screenings of both odorant receptors and the ligands as well as pharmacological cartelizations of a receptor-ligand pairs of interest. The main advantage of this technique is that we can assess odorant-to-receptor activation efficiently and reliably.
Heterologous odorant-to-receptor expression system using live cells. Begin by observing Hana3A cells under a phase-contrast microscope to ensure cell viability and to estimate confluence. Then aspirate all medium from the cell culture dish.
And wash the cells by adding 10 milliliters of phosphate buffered saline onto the plate, swirling the dish, and aspirating the PBS. Next, add the three milliliters of 0.5%trypsin-EDTA onto the plate. Leave the trypsin on the cells for one minute or until all cells are afloat.
Inactivate the trypsin by adding 5 milliliters of MEM with 10%FBS and pipetting up and down to break up chunks of cell mass. After performing a cell count, transfer 2x10 to the 6th cells per 96-well plate to be transfected to a 15 milliliter tube. It is important that you calculate the correct number of cells to be prepared onto 96-well plate to avoid over or undergrowth of cells prior to stimulation.
Centrifuge the tubes at 200 times G for five minutes. And then aspirate the supernatant without disturbing the cell pallet. Add six milliliters of medium without antibiotics for each 96-well plate and pipette up and down without creating air bubbles to break up chunks of the cell mass.
Transfer the suspended cells into a reservoir. Using a multi-channel pipette, pipette 50 microliters of cells into each well of the 96-well plates. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.
Prior to transfection observe the plated cells under a phase-contrast microscope to ensure a proper confluence of approximately 30 to 50%per well and then return to the incubator. Follow the row tagged odorant-to-receptor construct, the accessory factor constructs, and the cyclic AMP biosensor variant construct. Then prepare a plasma transfection mixture in 500 microliters of MEM for each 96-well plate.
Next, prepare a transfection mixture containing 18 microliters of lipid-mediated transfection reagent in 500 microliters of MEM. Mix the transfection mixture with the plasma mixture by pipetting up and down. Then incubate at room temperature for 15 minutes.
After the incubation, stop the reaction by adding 5 milliliters of MEM with 10%FBS to the transfection mix. Next, spread out a thick layer of sterile paper towels in the cell culture hood. Take a 96-well plate with cells from the incubator.
Gently and repeatedly, tap the plate upside down on the pile of paper towels so that the medium is completely absorbed by the paper towel. Transfer 50 microliters of the combined transfection mixture to each well of the 96-well plate and incubate overnight for 18 to 24 hours at 37 degrees Celsius with 5%carbon dioxide. You can manage the transfection and the stimulation schedule should persist on instrument for more than one 96-well plate is tested in one experiment so that all plates are mirrored after same transfection and distillers exposure interval.
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 heaps and five millimolar glucose to Hanks'balanced salt solution. Thaw 55 microliters alloquats of real-time cyclic AMP assay substrate reagent alloquats on ice.
Prepare 2%equal abration 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%equillibration solution into each well and incubate at room temperature in the dark for two hours. Prior to the end of the incubation time, dilute thawed odorant stock solutions to working concentration and 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.
379 unique human odorant receptors were screened against 30 micromolar muscone using the real-time cyclic AMP assay. Colored blocks along the X axis indicate different human odorant-receptor families. The Y axis represents normalized luminescence 30 minutes after odorant addition when the response reached the maximum for the positive control.
The response of OR5AN1 is indicated. This figure shows real-time measurements of OR5AN1 activation by different concentrations of muscone and the result from a no-odor negative control. The arrow indicates the time point of oderant addition.
Here, a concentration response curve of OR5AN1 against muscone 30 minutes after oderant addition is shown. The technique described here takes a total of three days starting with a stock plate of culture tiers on the first day. Once mastered, the process on the last day can be completed in three hours for each 96-well plate if it is performed properly.
After watching this video, you should have a good understanding of how to to manage the activation of oderant receptors using a cyclic AMP assay to harness the results.
