The overall goal of the following experiment is to identify functionally activating ligands of orphan G protein coupled receptors or G PCRs. This is achieved by transiently transecting, A-G-P-C-R of interest and cot transecting a promiscuous G alpha 16 construct in a cellular expression system to obtain temporary heterologous expression of both proteins. As a second step, the transfected cells are loaded with the calcium sensitive dye flu oh four, which detects the release of calcium from intracellular stores through an increase in fluorescent signal upon calcium interaction.
Next, the fluorescent based calcium mobilization assay is performed by challenging the heterologous expressed receptor with a compound library of candidate ligands to determine compounds that activate the receptor and elicit an intracellular signaling cascade resulting in the release of calcium from the endoplasmic reticulum, which can be measured as a fluorescent signal. Results are obtained that show a concentration response curve with the half maximal effective concentration of an activating ligand, which is based on the detection of different fluorescent signals depending on the administered ligand concentrations to the cellular expression system. The main advantage of performing transient transfect to obtain temporary expression of your GPCR over the use of stable clonal cell lines is that it does not require the time consuming establishment of such stably transfected cell lines cot.
Transfection of the promiscuous G alpha 16 subunit can redirect the intracellular signaling pathway of most GPCRs toward the release of calcium regardless of the native signaling pathway in endogenous settings. As such, this setup allows medium throughput screening of hundreds of compounds on GPCRs of interest in a short time span, demonstrating the procedure will be yellow gas, a PhD student and Nick Su, a technician from our laboratory Grow the HEK 2 93 T cells in T 75 flasks three days before the actual calcium mobilization assay takes place, passage the cells when a confluence of 80%is reached. Prepare one T 75 flask for transfection with the receptor construct and one for transfection with an empty expression vector as a negative control.
Incubate the flasks at 37 degrees Celsius in a 5%CO2 humidified incubator for 20 to 24 hours until the cell cultures approach 50 to 70%confluence as shown here. Next, add 3.9 micrograms of the receptor construct or empty vector and 3.9 micrograms of the G alpha 16 expression Construct to a 1.5 milliliter micro fuge tube. Add 500 microliters of the jet prime buffer.
Mix well by vortexing the tube followed by a short spin. Then add 37.5 microliters of the jet prime reagent vortex and spin down for one minute at maximum speed. Incubate the transfection mix for 10 minutes.
At room temperature, label one population of cells as transfected with the expression vector, encoding the GPCR of interest and the G alpha 16 construct and the second flask with the empty vector and the G alpha 16 construct. Following incubation, add the transfection mix dropwise to the cell culture medium, making sure to pipette directly into the medium and avoid contact with the walls of the culture Flask. Incubate the flasks at 37 degrees Celsius in a 5%CO2 humidified incubator for 20 to 24 hours for the calcium mobilization assay.
The transfected cells are collected and seeded into 96. Well black walled clear bottom plates. First coat the plates with 60 microliters per well of room temperature.
Phosphate buffered saline with fibronectin. Incubate the plates at room temperature for one hour with the lid on. Remove the solution from the wells and incubate the plates again for one hour without the lid at room temperature.
Meanwhile, take off the old growth medium from the cells. Rinse dead cells off with three milliliters of PBS and remove the PBS. Then add three milliliters of room temperature PBS tryin EDTA incubate for one minute before removing the solution.
At this point, the morphology of the cells changes from star shaped to sphere shaped. Loosen the cells from the bottom of the flask by gentle tapping and collect them by rinsing several times with 10 milliliters of room temperature. DM em transfer medium.
Transfer the cells into a 50 milliliter falcon tube. To count the number of cells per milliliter, add 100 microliters of the cell culture to a 1.5 milliliter micro centrifuge tube. Then add 100 microliters of lysis buffer and mix by tapping the tube.
Finally, add 100 microliters of stabilizing buffer and mix by tapping. Fill a nucleo cassette with the cell suspension and count the cells with the counter. The nucleo view software automatically multiplies the number of cells by three as the cells were diluted by a factor of three.
When adding the lysis and stabilizing buffer, dilute the cells to a final concentration of 600, 000 cells per milliliter and seed 150 microliters of the cells per well in the coated plates. To obtain a cell density of about 90, 000 cells per well avoid air bubbles and tap the plate to spread the cells evenly. In order to get a contiguous cell layer, incubate the plates at 37 degrees Celsius in a 5%CO2 humidified incubator for 16 to 24 hours.
Discard the medium from the cells. Wash the cells with 200 microliters of PBS per well and remove the PBS. Note that all the following steps that involve operations with the cells should be performed in the dark because from this point on, the cells will be loaded with the fluoro four.
Add 55 microliters of loading buffer per well wrap the plate in aluminum foil to prevent exposure of the plate to light and incubate for one hour at room temperature. Next, use wash buffer to solubilize the dried peptides in siliconized 1.5 milliliter micro centrifuge tubes. Prepare a dilution series to perform a concentration response analysis and determine the EC 50 value of a ligand.
Keep in mind that 50 microliters of a ligand from the compound plate will be transferred into a well with 100 microliters of wash buffer on the cell plate during the assay, so prepare the compounds at three times their desired final concentration. Next, add 70 microliters of the ligand in the corresponding well of the compound plate in triplicate. Include positive and negative controls on each plate.
Discard the loading buffer from the cell plate and add 100 microliters of wash buffer to each well preventing exposure to light. Incubate the plate for 15 minutes at room temperature. Discard the wash buffer from the cell plate and add 100 microliters of new wash buffer to each.
Well incubate the cell plate, the compound plate, and a tip rack for 15 minutes. At 37 degrees Celsius, create and load the desired protocol file with the software and activate the temperature control unit for the reading chamber here. Calcium responses are measured at 37 degrees Celsius for two minutes, one row at a time with a 1.52 second interval between successive readings at 525 nanometers.
Set the excitation of flu oh four to 488 nanometers and transfer a total volume of 50 microliters of compound to the cell plate. 18 seconds after the reading. Start with the dispense speed set to 26 microliters per second and a pipette height of 135 microliters.
Position the compound and cell plate and the tip rack in the appropriate drawers of the station device. Perform a single read of the cell plate at the same excitation and emission wavelength in the endpoint mode before starting the actual screen in flex mode. Start the assay and proceed to analyze the data using the software depicted.
Here are graphs corresponding to one of three replicas of the doofus short NPF one concentration series. The negative control did not induce a fluorescent signal. While the positive control elicited strong activation of the endogenous protease activated receptor one leading to a high fluorescent signal.
The software allows entering of formulas to determine the percentage of activation or the standard errors of the different concentrations among others. The resulting data are used to compose preliminary concentration response curves to estimate the EC 50 values as shown for the four esophagus short NPF peptides. The curves also include the data for the negative control, where the concentration series of the drosophila short NPF peptides were tested on cells transfected with an empty vector.
The results indicate that the addition of the peptides to these cells have no effect on endogenous receptors, but indeed activate the receptor of interest. Preliminary concentration response curves of the fluorescence based calcium mobilization assay were performed and the tested concentrations were adapted to cover the dynamic range of the curve. The EC 50 values of drosophila short npf 1, 2, 3, and four are similar, indicating that they are equally potent to activate the receptor Following this procedure.
Other methods like structure activity, relationship studies can be performed to answer additional questions such as which amino acids of the peptide are crucial for receptor activation. After watching this video, you should have a good understanding of how to perform a fluer sense based calcium mobilization as a for identifying functionally activating ligands of G protein coupled receptors. This includes the maintenance of solar expression systems followed by the transgender transfection of the cell system by receptor of interest.
After rigid calcium mobilization assay can take place.
