Rhodopsin misfolding causes progressive retinal degeneration called retinitis pigmentosa. While a traditional imaging method have limited capacity to quantify the rhodopsin transport, this newly-developed image-based assay allows for high throughput screening, eliminating false positives. This technique allows for a quick evaluation of drug pharmacodynamics.
In the eye of rescuing the folding of disease-causing rhodopsin, thus accelerating the potential for a treatment for the quietly untreatable and blinding disease, retinitis pigmentosa. The cellular localization of a membrane protein is very important for its function. This method can be easily modified and applied to quantify the transport of any other membrane protein of interest.
Make sure you prepare a plate map and gently add and aspirate liquid from each well of the plate, to keep cell number and the immuno state conditions consistent between the cells. This will yield a high Z factor, and reliable results. To begin, seed 5000 cells per well into a black walled, clear bottom, poly lysine treated 384-well-plate.
Incubate the plate at 37 degrees Celsius with five percent CO2 for three hours for the cells to attach to the bottom of the plate. Next, use a multichannel pipette to add 10 microliters per well of 5X working solutions, according to a pre-determined plate map like the one shown here. Now, add 10 microliters per well of 2%DMSO to columns 22 and 24.
Also, add 10 microliters per well of 25 micromolar 9-cis-retinal to column 23 in a dark room under dim red light. When finished loading all of the various test compounds, cover the plate with aluminum foil, and incubate at 37 degree Celsius for 24 hours. Take out the 384-well-plate in a dark room with dim red light.
Here, using 8-channel-aspirator, connected to a vacuum collection bottle, to gentle aspirate the medium from the wells of the plate. Next, use a multichannel pipette to add 20 microliters per well of freshly prepared 4%Paraformaldehyde to the entire 384-well-plate, and fix the cells for 20 minutes at room temperature. After fixation, place the plate into normal light.
There, use an 8-channel-aspirator, to aspirate the Paraformaldehyde in each well, and use a multichannel pipette to add 50 microliters per well of PBS. Aspirate and replace the PBS for a total of three wash cycles. Then, add 20 microliters per well of 5%goat serum to each well, and incubate the plate at room temperature for 30 minutes.
Following incubation, aspirate the wells, and add 15 microliters of 20 micrograms per milliliter B630 anti-rhodopsin antibody, in 1%goat serum to the wells in rows A to O.Next, add 15 microliters per well of 1%goat serum to row P for the secondary antibody only control group. Incubate the plate at room temperature for 90 minutes, or at four degrees Celsius over night. Then, cover the plate with aluminum foil to avoid photobleaching of the fluorophores.
Next, wash the plate three times with 50 microliters per well of PBS. Following the last wash, aspirate the PBS, and add 15 microliters per well of five micrograms per milliliter of Cy3-conjugated goat anti-mouse IgG antibody. Cover the plate with aluminum foil, and incubate the samples at room temperature for one hour.
Following the incubation, wash the plate three times. Then, add 50 microliters per well of PBS, containing one microgram per milliliter of Hoechst stain at room temperature for 15 minutes. Finally, seal the well plate with a transparent film, cover it with aluminum foil, and store at four degrees Celsius for up to one week.
Remove the foil and place the sample plate into a high content imager, with A1 positioned on the top left corner of the plate. Next, open the image acquisition software to set up parameters for image acquisition. Open the plate acquisition setup window and create new setting or load an existing setup file.
Select the 20X objective and set up pixel binning as two, so the calibrated pixel size is 0.80 by 0.80 microns. Then, set the scan lines as 2000, and the image size as 1000 by 1000 pixels per site. Next, select the plate type to be imaged.
Use information provided by the manufacturer to fill in the plate dimensions. Now, select the wells to be imaged along with four sites to be imaged per well. Avoid the side of the well, which are touched by the aspiration tips.
For imaging, select excitation lasers as 405, 488 and 461 nanometers. Then, select the emission filters for DAPI, FITC and Texas Red channels. Optimize the laser power and gains of each channel, to ensure, the positive control wells are not saturated.
Select well to well focus for auto-focus. Then, select the initial well to be acquired, and set the site auto-focus for all sites. Also, select four averages per line for each channel, and optimize the Z-offset value for each channel.
Validate, that everything is properly set up, by first testing two to three wells at the corners of the plate, to make sure the images are on focus for all tested wells. Next, check, that images from all sites per well have cells with more than 40%confluence. Finally, check, that the fluorescence intensities in all of the channels are all about half saturated in the 100%control wells.
Then, save the image acquisition method, and run the entire plate. Watch the imager until it finishes capturing images from the first column of the plate to double-check the image quality before leaving the imager. When finished, remove the plate and store it at four degrees Celsius for future use.
Pull out image data, using a high content image analysis software. Select one of the 100%control wells in column 23 to set up the parameters. First, select the multi wavelength cell scoring as the analysis method, and start configuring the additional settings.
Define the nuclei using images from the DAPI channel. Preview, to make sure the defined nuclei shapes in the selected well fits well with the nuclei images. Next, define the shape of cell in the FITC channel, where Rhodopsin Venus is imaged.
To accomplish this, set up minimum and maximum diameters of each cell, minimum fluorescence intensity above background, as well as minimum area of each cell. Now, define the rhodopsin cell surface stain areas in the Texas Red channel, by setting up the minimum and maximum diameters of cell shape, minium fluorescence intensity above background, as well as minimum area of each stained cell. Test the current algorithm in five wells to determine, if the settings are optimized.
Then, save the settings and close the configuration window. Run all the wells with the optimized analysis method. When finished, export the object number as the intact cell number.
Export the average intensity of the FITC channel as the Rhodopsin Venus intensity, and export the average intensity of the Texas Red Channel as Rhodopsin intensity on the cell surface. Use the spreadsheet software to generate a two-color heat map for each parameter. Arrange the name of cell lines on the x-axis, and the compounds on the y-axis.
Here, our images of P23H rhodopsin Venus treated U2OS cells, expressing Venus fluorescence in green, and cell surface immuno-staining in red. Cells were either treated with DMSO or five micromolar 9-cis-retinal. The rhodopsin transport assay, comparing the rhodopsin amount in the whole cell under various conditions, shows a significant recovery in the P23H mutant cell line, when treated with 9-cis-retinal.
In addition, the rhodopsin intensity on the cell surface, and the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity in the whole cell, are not significantly lower for the P23H mutant, compared to the wildtype rhodopsin treated with DMSO. A heat map is an efficient way to compare the average rhodopsin amount and localization per cell across multiple mutants. Here, wildtype controls and six rhodopsin mutants are listed horizontally, and the effects of compound treatments are compared vertically.
In agreement with previous studies, the rhodopsin intensity on the cell surface and the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity are lower for the six mutants compared to the wildtype treated with DMSO. Compounds one, two, six and nine significantly increased the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity in T4R, P23H, D190N, and P267L rhodopsin mutants, suggesting that these compounds rescue the transport of these rhodopsin mutants to the plasma membrane. Keep in mind, that cells need to be between 50 and 70%confluent before fixation, as this is critical for image analysis.
Also, we showed images of a wells across the whole plate. Make sure, that images in focus, and that the fluorescence, that don't exit, have all the threshold value for any channel. Up to a selecting compounds, that rescue misfolded rhodopsin transport, we can validate the process, then pass the efficacy of these compounds in vivo, using a mouse-model expressing the rhodopsin P23H mutant.
This method allows us to quantify membrane protein amount, and its localization. Therefore, it is a great tool for micro mistake started on membrane protein per assist save and degradation. Paraformaldehyde, as is in this experiment, has procedures involving the handling of this reagent.
It will be done under the hood. Waste including this reagent should be properly disposed of as a hazardous chemical.
