The overall goal of this live cell Forster resonance energy transfer, or FRET based assay, is to assess G protein-selective G protein coupled receptor or GPCR conformations under different agonist conditions. This method can help answer key questions in the GPCR field about GPCR interactions with different effectors and the effect of different drugs on receptor confirmation. The main advantage of this technique is that the sensors are modular, and can be easily re-engineered for different GPCR and G alpha peptide combinations, including the full and G alpha sub unit.
Though this method can provide insight into G protein selection and response to different lygens. It may also be applied to explore the physiological consequence of differential G protein activation. Before beginning the procedure, culture the cells of interest in complete medium at 37 degrees Celsius in a humidified atmosphere at five percent carbon dioxide until the cultures reach confluency.
Next, detach the cells with Trypsin and passage the cultures at eight times 10 to the fifth cells per cell in two millimeters of medium into tissue culture treated six well dishes for sixteen to 20 hours of culture. After 16 to 20 hours, prepare one transfection reaction per well when the cells have adhered to the plates in a biological safety cabinet and allow the reagent mixtures to equilibrate for 15 to 30 minutes. At the end of the incubation, add one reaction to each cell culture drop-wise across the well.
Gently shaking the plate to ensure a thorough distribution of the reagent throughout the cultures. Then return the plate to the cell culture incubator. After 20 hours, analyze the cultures for protein expression of the plasma membrane and assess cell health.
If substantial internalization of the protein is observed, monitor the transfection until a significant expression is detected at the plasma membrane. Assessing the protein expression level is imperative for an optimal experimental setup. If the cells transfected poorly or the protein expression is too low, wait to harvest the cells.
Otherwise, the fluorescent signal may be inadequate for analysis. When the cells are ready to be harvested, transfer them to the bio-safety cabinet and gently remove one milliliter of medium. Using a P1000 pipette, resuspend the cells in one milliliter of remaining medium per well and transfer the cell suspensions into individual 1.5 milliliter microcentrifuge tubes.
Count the cells and pellet the cells by centrifugation. Remove the medium from the cell pellet. Gently resuspend the supernatant in one milliliter of 37 degrees Celsius cell buffer and repeat the centrifugation.
Resuspend the pellet at a four times 10 to the sixth cell per milliliter concentration in fresh cell buffer after the second spin. Next, add 90 microliters of control cells to a cuvette and acquire the control FRET spectrum. After collecting three to five repeat control spectra with a fresh 90 microliters of cells for each measurement, repeat the suspension, spin and wash steps for transfected cells.
Aliquot 90 microliters of transfected cells to each 500 microliter tube in holder two to six and eight to 12 in a heat block, gently resuspending the cells between each aliquot. When all of the cells have been transferred, add 10 microliters of drug buffer to tubes two to six for the untreated condition samples. Then add 10 microliters of the one millimolar of the drug solution to tube eight.
Start a timer to countdown from 10 minutes and use a P200 pipette to gently mix the contents of the tube. Close the tube after mixing and return it to the heat block. Then immediately pick up tube two.
Mix its contents gently with a new tip. Add 90 microliters of the transfected cell suspension to the untreated condition cuvette and place the cuvette in the fluorometer. Acquire the FRET spectrum for the sample using the same parameters as for the control spectra.
At nine minutes and 10 seconds, spike tube nine with 10 microliters of the one millimolar drug solution. Gently mix the tube contents and return the tube to the heat block. Then immediately pick up tube three and mix and measure the cuvette contents as just demonstrated for tube two.
At five minutes and 10 seconds, gently mix tube eight with a P200 pipette. Add 90 microliters of the cell suspension to a separate cuvette for the drug treated sample measurement and acquire the FRET spectrum for the drug condition sample. After all of the spectra have been acquired, save the project files.
Thoroughly wash the cuvettes with ultra pure water and restock the tubes for the next condition. In this image, a homogeneous cell culture monolayer that is optimal for six well plating and transfection is shown. These cells growing in clumped patterns that lead to dendritic shapes however are not recommended for a consistent plating and accurate protein expression analysis or FRET measurement.
The transfection conditions can also be optimized to achieve reproducible expression. Once all of the data has been collected and entered into a CSV file for analysis, the generated results will resemble these representative RAW and Normalized mean FRET spectra. All of the RAW FRET spectra from this representative experiment demonstrate a smooth, sufficient signal to noise range allowing a consistent data analysis with a minimal water peak from the sample.
When the data is normalized at 475 nanometers, the counts per second of this value is set to 1.0 and the significant change at the 525 nanometer reading between the treated and untreated samples becomes apparent. If the protein expression is low or there is a poor transfection efficiency or low cell density in the cuvette for the flourescence reading, the RAW data may be noisy causing an issue in the background subtraction and normalization resulting in spectra that do not align and cannot be analyzed. Once mastered, this technique can be completed in 25 minutes if it is performed properly.
While attempting this procedure, it's important to remember to be gentle when handling the cells throughout all steps of the experiment. Following this procedure, the technique can be expanded to test different receptor FRET sensors, different drugs and effectors and different cell types. After its development, this technique has contributed to the GPCR field where recent studies using these sensors have shed light onto the molecular mechanism of G protein selection.
After watching this video, you should have a good understanding of how to assess specific GPCR confirmations using a live cell tether FRET construct measurement assay. Don't forget that working with mammalian cell culture and certain experimental drugs can be extremely hazardous and that precautions such as wearing the appropriate personal protection equipment should always be taken while performing this procedure.
