The overall goal of the following experiment is to perform a high throughput measurement of olfactory receptor activation via a luciferase assay. This is achieved by transecting H three A cells with vectors containing accessory proteins, firefly and vanilla luciferase and olfactory receptors in order to express a different olfactory receptor. In each well of a 96 well plate.
As a second step, the cells are stimulated with odors. Next luciferase substrate is added causing the cells that produced luciferase enzyme to luminesce, thus functioning as a reporter for olfactory receptor activation. Ultimately, results show the responses of the population of olfactory receptors to the odors based on the luminescence of each well in the plate.
The main advantages of this technique over existing methods such as adenoviral, transduction of native neurons or calcium imaging, is that this technique both scales to high throughput screens and utilizes the native CM mediated second messenger pathway to examine olfactory receptor activation. This method can help answer key questions in the field of olfaction, such as matching odor ligands, so olfactory receptors, and examining the effect of genetic variation on olfactory receptor activation. The Hanna three A cells used in this protocol are maintained in M 10 media at 37 degrees Celsius with 5%CO2, and should be 100%confluent in order to plate cells for transfection aspirate media from a 10 centimeter dish of cells wash cells.
By adding 10 milliliters of PBS swirling the dish and aspirating the PBS add three milliliters of 0.05%tripsin EDTA and wait for about one minute for the cells to dissociate at room temperature. Inactivate trypsin by adding five milliliters of M 10 media break up cell clumps by tri rating roughly 10 times with a 10 milliliter pipette. Pipette carefully to avoid introducing air bubbles into the media.
For each 96 well plate transfer one milliliter of cells into a 15 milliliter conical tube. Centrifuge at 200 times gravity for five minutes and aspirate the supernatant without disturbing the cell pellet res. Suspend the cells in 12 milliliters of M 10 pipette 50 microliters of cells to each well of the 96 well plates and incubate overnight at 37 degrees Celsius with 5%CO2.
Prior to starting this procedure, observe the cells plated on the previous day to ensure a proper co fluency of approximately 30 to 50%per well. Return the plates to the incubator and proceed. To prepare the transfection, mix the plasmids for expressing accessory proteins.
Firefly luciferase and ran vanilla luciferase have each been diluted to a concentration of 100 nanograms per microliter in TE buffer to make the plasmid mix pipette the plasmids into mem medium per the volumes indicated in this table. For each 96 well plate dilute 18 microliters of lipid transfection reagent in 450 microliters of mem medium to make the lipid transfection mix. Next, pipette the plasmid.
Mix the plasmid with the opsin tagged olfactory receptor and the lipid transfection mix to a clean MICROTITER plate. To make the complex detailed in this table, the well plus 10%calculation is important to ensure sufficient volume for subsequent steps. Mix the solution by tri curation and incubate at room temperature for 15 minutes.
Stop the reaction by adding M 10 according to the table. For the success of this procedure, it's important that cells are never left without media for longer than two minutes. While the solutions are being added to the wells.
Remove the cells from the incubator and invert the cell plate and tap out the media pipette 50 microliters of complex to each well and incubate overnight at 37 degrees Celsius with 5%CO2 on the day after transfection of olfactory receptors. Observe the transfected cells to ensure a proper co fluency of 50 to 80%per well return the cells to the incubator. The odor has been diluted to a stock concentration of one molar and DMSO for screening experiments dilute each stock solution of odor to 100 micromolar.
In addition, prepare a no odor control containing only CD 2 93 medium to control for olfactory receptor background activation. Remove the cells from the incubator and tap out the media from the cell plates. Pipette 25 microliters of odor stimulation solution to each well and incubate at 37 degrees Celsius with 5%CO2 for four hours.
Begin this procedure. Thawing one milliliter of firefly luciferase substrate per 96. Well plate prepare fresh firefly luciferase reaction quencher and ran vanilla luciferase substrate reagent.
Approximately one milliliter of reagent is needed per 96 weld plate. Next, prepare the luminescent microplate reader within the system icon under the preheating tab. Check the box for on and set the temperature of the machine to 25 degrees Celsius under the dispenser tab.
Prime each dispenser with 1000 microliters of 70%ethanol, followed by 1000 microliters of distilled water. Use separate aliquots of alcohol and water for each dispenser. Prime each dispenser with 1, 500 microliters of air prime dispenser.
One with 1080 microliters of firefly luciferase substrate prime dispenser. Two with ranil luciferase substrate. Be careful not to cross contaminate the luciferase substrates.
Set up the following protocol to read firefly luciferase and vanilla luciferase luminescence within the software. Under the file menu. Click on new task highlight protocols and click on create new.
In the next window, the circle next to standard protocol should be selected. Click okay. Double click on procedure.
On the left hand side of the screen dispense 10 microliters of firefly luciferase substrate to all wells using dispenser one under the actions menu, click dispense in the dispen step window. Set dispenser to one. Priming to none.
Dispense volume to 10 microliters and rate to 225 microliters per second click okay. Shake the plate for 30 seconds under the actions menu, click shake in the shake step window. Set intensity to medium and duration to 30 click.Okay.
Read the luminescence of all wells for 0.5 seconds per well under the actions menu. Click read in the read step window set detection method to luminescence read type to endpoint integration time to 50 filter sets to one emission to whole optics position to top gain to 1 35 and read height to 1.00 millimeters. Click okay.
Dispense 10 microliters of ran luciferase substrate to all wells using dispenser two. Set the conditions as shown for using dispenser. One except set dispenser to two.
Shake the plate for 30 seconds. Read the luminescence of all the wells for 0.5 seconds per well. Remove the lid from the 96 well plate and place the plate in the Microplate reader.
Start the previously set up program to read plate luminescence after reading is complete. Clean the reagent dispenser pumps from the system icon under the dispenser tab. Purge 1000 microliters of firefly luciferase substrate from the firefly luciferase dispenser into a recovery tube prime.
Each dispenser with 1000 microliters of distilled water followed by 1000 microliters of 70%ethanol. And finally, 1, 500 microliters of air shown here are results from a primary screen that tested 328 olfactory receptors or ORs against 26 odors at a concentration of 100 micromolar. This histogram depicts the frequency or count of baseline luciferase values calculated for each odorant or pair in the screen as odorant receptor activation pairs are sparse.
The majority of the values are centered at zero and the large central distribution estimates the noise distribution for this assay. Dose response curves were performed on 48 odorant or pairs randomly distributed across the range of baseline values as indicated by the colored bars. Blue bars are pairs that were classified as agonists based on the full dose response, and red bars are pairs that were not classified as agonists.
True positive rate was then plotted against the false positive rate using the R statistical package. The area under the curve or a UC is 0.68 indicating that odorant or pairs with higher luciferase screen values are more likely to pass dose response than those with lower values. These data suggest that the primary screen is a useful method to enrich for odorant or pairs that will be classified as agonists in a full dose response experiment.
Once mastered, this technique can be done in approximately six hours for 5 96 ole plates, excluding the incubation time if it's performed properly. After watching this video, you should have a good understanding of how to perform a high throughput analysis of olfactory receptors using transfection odor stimulation, and measurement of activation with luciferase assay.
