Our protocol enables to measure detection and discrimination of volatile odors with a panel of odorant receptors. This method opens up the possibility to develop odorant receptor-based biosensors. This technique allows real-time in vitro monitoring of odorant receptor activation upon odorant molecules in vapor phase.
It fills gap between in vitro and in vivo approaches. This method can be used to understand the kinetics of events that lead to odor perception, including the role of mucosal metabolic enzymes. To begin, prepare M10 and M10 PSF according to the manuscript.
Cells are cultured in 10 milliliters of M10 PSF at 37 degrees Celsius and 5%carbon dioxide. Observe the culture dish under a phase contrast microscope. When it reaches 100%confluence, split the cells to a lower concentration by aspirating the media and washing the cells gently with 10 milliliters of PBS.
Then aspirate the PBS and add three milliliters of trypsin-EDTA. Let it react for approximately one minute until the cells dissociate from the dish. Add five milliliters of M10 to inactivate the trypsin, and pipette up and down to detach the cells attached to the dish.
Transfer the eight milliliters of cell suspension from the dish to a 15-milliliter tube, and centrifuge the tube at 200-times-G for five minutes. Aspirate the supernatant and resuspend the cells in five milliliters of M10 PSF by pipetting up and down to break up any cell mass. Avoid creating bubbles in the tube.
To obtain 20%confluence of cells, add nine milliliters of M10 PSF in a new 1, 000-millimeter cell culture dish, and transfer one milliliter of the resuspended cell solution. Incubate at 37 degrees Celsius and 5%carbon dioxide. This dish, seated at 20%confluence, will reach 100%confluence in 48 hours.
Place the dish under a phase contrast microscope to observe. Take 1/10 of the 100%cell confluence culture dish. Aspirate the media and wash the cells gently with 10 milliliters of PBS.
Then treat the cells with trypsin-EDTA and M10, centrifuge, and treat again with M10 PSF as previously performed. To realize one 96-well plate, add 500 microliters from the five milliliters of resuspended cells to 5.5 milliliters of fresh M10 PSF. Mix the cells and M10 PSF without generating air bubbles.
Pipette 50 microliters of the diluted suspended cells into each well of the 96-well plate using a multichannel pipette. Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. First, place the 96-well plate cultured overnight under a phase contrast microscope, observe the cell confluence, and make sure it is between 30%and 50%Prepare a first transfection mix in a 1.5-milliliter tube that contains the plasmids common to the entire plate according to the manuscript.
Rho-pCi is an empty-vector negative control, and Olfr1377 is a positive control known to respond to the tested odorant acetophenone. Split the mix into two transfection mixes containing one of the control plasmids. Prepare a second transfection mix containing 500 microliters of MEM and 20 microliters of lipofectamine 2000 reagent.
Add half of the second mix to each reservoir well and gently mix by pipetting up and down. Incubate the tube for 15 minutes at room temperature. Add five milliliters of M10 for one 96-well plate.
Add 2.5 milliliters into each reservoir well and mix gently. Replace the M10 PSF in the previously plated 96-well plate with 50 microliters of the final transfection media. Incubate overnight at 37 degrees Celsius and 5%carbon dioxide.
Then open the door of the luminometer and insert the tube connected to the vacuum pump. Vacuum the chamber of the luminometer overnight. After transfection, observe the 96-well plate under a phase contrast microscope to assure a cell confluence between 60%and 100%Prepare the stimulation solution of Hank's Balanced Salt Solution containing 10-millimolar of HEPES and five-millimolar of D-glucose.
Remove the transfection medium from the 96-well plate and wash the cells by adding 50 microliters of fresh stimulation solution to each well. To dilute the GloSensor cAMP reagent solution, pipette 2.75 milliliters of the stimulation solution into a five-milliliter tube and 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. After the luminescence measurement, remove the remaining odorant inside the luminometer by vacuuming odorants in the reading chamber extensively for at least two hours.
Replace with fresh air by sending compressed air for five minutes before incubating the next odorant to avoid cross-contamination of odor volatiles. To begin data analysis, export the data from the luminometer software. Average the replicates of the same OR for each recording time.
To calculate the normalized OR response to any eventual control, divide the empty vector-averaged value to the OR-averaged value at each recording time. Normalize each OR response to their basal activity by dividing the averaged OR response at zero seconds to each recording time response. To obtain a single OR response value for the curve of each OR, sum all the luminescence values of each recording time for each OR in order to obtain the area under the curve.
In this experiment, the real-time activation of mice ORs upon vapor odorant stimulus was monitored over 20 measurement cycles. An empty vector control was used to assure that the odorant-induced activities of the tested ORs were specific. The data for each well were first normalized to the empty vector control-averaged value for each cycle.
Then, to compare different ORs'responses, the data were normalized to the OR levels of basal activity in this assay. Single-activation values for each OR were computed by calculating the area under the curve for each OR by summing the emission values of all measurement cycles. Additionally, dose-dependent responses can be measured using increasing odorant doses.
The response of Olfr1377 to acetophenone stimulation at five concentrations was recorded. Only the three higher concentrations were able to activate Olfr1377. The pure compound stimulation shows a tendency to decrease the OR response over time, likely due to cell toxicity.
During the step, you should add the diluted odorant between the well, and be prepared to start quickly the recording of the plate. The time between odorant addition and monitoring should be minimized and constant between two experiments. In atria cells are biohazardous material, so remind to dispose the cells in the appropriate dispensaries.
