The overall goal of the following experiment is to detect modulation of G-protein coupled receptors by potassium channels, utilizing label free resonance wavelength grading optical biosensors to achieve this unresected, HEK 2 93 cells and HEK 2 93 cells stably expressing rat sodium activated potassium channel slack B are seated onto a 384 well optical biosensor plate and allowed to incubate overnight. Next, a baseline measurement of mass near the plasma membrane is performed and agonists of endogenous G-protein coupled receptors or G PCRs are added. As a final step, the change in signal resulting from GPCR activation is measured and the potassium channel modulation of this response is calculated.
Ultimately, this method can help answer key questions in the fields of neuroscience and cellular signaling by quantitatively measuring signaling mechanisms between G-protein coupled receptors and potassium channels. The main advantages of using this technique over the methods that currently exist to look at the interactions between channels and proteins, for example, CO and precipitation assays are that the assay can be carried out using living cells. It doesn't require the use of exogenous labels, which could alter the structure of the interacting protein.
And finally, within the living cells, you can actually follow the interactions in real time. Though this method can provide insight into signaling between GPCRs and potassium channels. It can also be used to study cellular responses resulting from ligand induced changes in cell adhesion and spreading toxicity, proliferation, migration, and any changes in the proximity of the plasma membrane to the optical biosensor.
The technique also has the potential to be used in discovery of new therapeutic approaches to diseases caused by ion channels or receptor abnormalities, such as many channelopathies because it can be used as a high throughput method to test the effects of pharmacological agents on channel protein. Protein interactions. We first had the idea to use this method when we realized it was necessary to develop an assay that could look at channel protein interactions in living cells.
Begin 384. Well optical biosensor plate coating by hydrating a titanium dioxide plate with 20 microliters per well of sterile tissue culture, grade water for 15 minutes. Then use a 16 channel pipetter to wash the plate one time with 50 microliters of phosphate buffered saline or PBS per well.
Next, add 20 microliters of a prepared fibronectin working solution to each well before incubating for two hours at room temperature to create the extracellular matrix or ECM following incubation. Wash the plate once with 50 microliters of PBS per. Well then add 40 microliters of a prepared ovalbumin working solution to each.
Well incubate the plate overnight at four degrees Celsius to block the plate after ovalbumin binding. Wash the plate three times with 50 microliters of PBS per well. ECM coated plates can be stored at four degrees Celsius for up to four eight hours.
Remove growth medium from both unresected HEK 2 93 cells and HEK 2 93 cells stably expressing slack B that has been cultured as described in the text protocol. Then add tripsin to dislodged the cells from the plate. Collect the cells in centrifuge at 1000 times gravity for one minute.
Remove the tripsin medium and resuspend the cells in growth medium. Using a hemo cytometer or an automated cell counter, determine the concentration of the cells to minimize the effects of evaporation during incubation dilute cells to achieve the desired number of cells per well. In a volume of 50 microliters of growth medium for these experiments, 1000 cells per well.
Were plated onto the biosensor after pipetting 50 microliters of the diluted cells to each, well allow the seated cells to incubate overnight in growth medium at 37 degrees Celsius, 5%CO2. To prepare the resonance wavelength grading or RWG optical biosensor assay. Remove the growth medium from the cells following incubation.
Wash the cells once with hank's balanced salt solution and then add 25 microliters of the solution to each. Well place the biosensor plate on the bind scanner and allow the cells to equilibrate to room temperature for one to two hours. Meanwhile, prepare a separate compound plate from which ligands of interest will be added to the biosensor plate during the assay.
For these assays, 25 microliters of solution containing compound was added to each well on the biosensor plate for a total volume of 50 microliters. To perform the RWG optical biosensor assay, take a baseline measurement with the scanner system for well's, A one through P 12, which represent half of the 384. Well plate use a maximum resolution of 3.75 microns per pixel for the central 1.5 millimeters of each well using the bind scan software, this baseline scan will take approximately 30 minutes.
Then add 25 microliters of the compound solutions to each well for wells, a one through P 12. Next, take a baseline measurement of wells A 13 through P 24. Following the baseline of these wells, add 25 microliters of the compound solutions to each of wells, A 13 through P 24.
As a final step, take a post compound edition measurement for wells, A one through P 12 and repeat for wells a 13 through P 24. To analyze the assay data, open the bind assay data for the baseline measurement in windows, and click the grid editor button to enable cell selection. The software will calculate metrics of interest defined by the user, open the plugins, which include the cell finder and baseline or function set parameters in cell finder so that cells are distinguishable from the background of the biosensor.
Export the cell map file, which contains the cell finder data for the baseline measurement. Repeat these steps for post compound addition data. Then open the baseline or plugin and import the cell map from the baseline measurement.
The software will calculate the shift in peak wavelength value or PWV between the background and post compound data. Select delta cell mean for the output of the mean shift in PWV for cells. The data can then be exported to Microsoft Excel for further analysis.
Slack b stably transfected, HEK 2 93. Cells and control unresected HEK 2 93 cells were seated at 1000 cells per well on 384. Well, ECM coated RWG biosensor plates, density gradient maps of mass were generated pre and 30 minutes post compound edition with the bind scan.
Software areas shown in red represent pixels identified as belonging to cells and yellow lines, distinguished cells from the gray plate background. The bind view software was utilized to determine the shift in PWV upon GPCR agonist edition by subtracting the PWV post compound edition from the baseline PWV. This is shown by a color gradient where red represents a positive shift in peak wavelength value and blue represents no change.
The endogenous M1 muscarinic receptor against carbahol was applied to both unresected control HEK 2 93 cells and HEK 2 93 cells stably expressing slack B and resulted in a positive increase in peak wavelength value compared to the control cells. The slack B transfected cells had a greater increase in peak wavelength value. S-F-L-L-R NH two, tri fluoro acetate salt and end terminal thrombin receptor activating peptide pentapeptide that exhibits agonist activity against the endogenous GPCR protease activated receptor one also showed differential responses in unresected and slack B transfected cells.
These results show the presence of the Slack B channel significantly increases the shift in PWV that occurs upon activation of endogenous G PCR in he K cells, and demonstrate the feasibility of using this assay to study channel protein interactions. After watching this video, you should have a good understanding of how to develop and perform a label-free optical biosensor assay. Once mastered, this technique can be performed over the course of two days if the cells and reagents have been prepared with care While attempting this procedure.
It's important to remember that assay parameters, such as the number of cells to be seated, the incubation time of ligands, et cetera, must be optimized for different cell lines. Once you've carried out this procedure, you can use other methods such as R and AI to try and determine which particular proteins are required for the observed change in the signal. Don't forget that working with human cell cultures can be extremely hazardous, and precautions such as working in a biosafety hood should always be taken while performing this procedure.
