We thank Dr. Mark E. Schurdak and University of Pittsburgh Drug Discovery Institute for providing the high-content imager and initial trainings. Dr. Krzysztof Palczewski (Case Western Reserve University) generously shared the 1D4 and B630 anti-rhodopsin antibodies. The plasmid containing the cDNA of mouse rhodopsin-Venus construct was shared by Dr. Nevin Lambert (Augusta University). This work was supported by the National Institute of Health grant EY024992 to YC and P30EY008098 from University of Pittsburgh Vision Research Core grant.

NOTE: The rhodopsin transport assay. 
1. Preparation and Culture of Cells
<ol>
	<li>Revive cryo-preserved U2OS stable cells expressing the wild type (WT) or mutant mouse rhodopsin-Venus fusion proteins. Thaw the cells at 37 &#176;C until only small ice crystals are left in the vial. 
	NOTE: The U2OS cells are used in this protocol because there is no photoreceptor cell line available for in vitro studies and the pre-ciliary biosynthesis of rhodopsin is regulated by similar molecular mechanisms in mammalian cells. Additionally, the U2OS cells attach tightly to the bottom of the plate and have large cell bodies, so they are ideal for the rhodopsin transport assay. Seven U2OS stable cells expressing the WT, T4R, P23H, P53R, C110Y, D190N and P267L rhodopsin mutants are used here as an example to show the quality and reliability of the assay. Stable cells expressing other rhodopsin mutants or other membrane proteins can be used, depending on the goal of the assay. Stable cells expressing human rhodopsin can be used in this assay if available. The mouse rhodopsin was used here because mouse and human rhodopsin proteins share 95% homology, and the compounds identified by this assay will be tested in vivo using a mouse adRP model carrying the rhodopsin P23H mutation8. The stable cells were generated as described previously19. The WT and mutant rhodopsin transcripts were quantified by q-PCR, and the clones of stable cells expressing similar levels of WT and mutant rhodopsin transcripts were selected for this assay. The expression of rhodopsin proteins was confirmed by immunoblot and immunostaining. The passage of cells used in this assay should be lower than 20.</li>
	<li>Transfer each line of cells into a 15 mL conical tube containing 10 mL of growth medium (high glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS) and 5 &#181;g/mL Plasmocin).</li>
	<li>Centrifuge the tube at 200 x g for 5 min. Discard the supernatant and resuspend the cell pellet with 5 mL of cell growth medium for each cell line. Transfer each line of cell suspension into a 60 mm tissue culture dish and incubate at 37 &#176;C with 5% CO2.</li>
	<li>Subculture the cells when the tissue culture dish reaches 90% confluence as described in reference12.</li>
</ol>
2. Seeding Cells at 5,000 cells per Well
NOTE: Perform the following procedures in a tissue culture hood.
<ol>
	<li>Treat the plate with poly-lysine.
	<ol>
		<li>One day before seeding the cells, treat a black-wall and clear bottom 384-well plate with 20 &#181;L/well of poly-lysine solution for 30 min.</li>
		<li>Aspirate the liquid and allow all wells to dry for 1 h. Put back the plate lid and store the plate at 4 &#176;C overnight for cell seeding.</li>
	</ol>
	</li>
	<li>Prepare the cell suspension.
	<ol>
		<li>Culture each cell line in growth medium in a 100 mm tissue culture dish until 90% confluence.</li>
		<li>Wash each dish with phosphate buffered saline (PBS) and detach the cells with 1 mL of 0.05% Trypsin at 37 &#176;C.</li>
		<li>Resuspend each line of cells in 10 mL of assay medium (high-glucose DMEM with 10% FBS, 100 units/mL penicillin and 100 &#956;g/mL streptomycin).</li>
		<li>Count the cells and dilute each line of cells to 1.25 x 105&#160;cells/mL with the assay medium.</li>
	</ol>
	</li>
	<li>Seed the cells.
	<ol>
		<li>Use an electronic multichannel pipette or an automated reagent dispenser to dispense 40 &#181;L/well of the cell suspension to three columns per cell line and fill columns 1-21 of the 384-well plate (Figure 2).</li>
		<li>Add 40 &#181;L/well of U2OS (P23H-Rhodopsin-Venus) to columns 22-23 and 40 &#181;L/well of U2OS (Rhodopsin-Venus) to column 24, as controls.</li>
		<li>Centrifuge the plate at 300 x g for 30 s to bring down all cells to the bottom of the 384-well plate. 
		NOTE: Avoid physical shock to the plate after cell seeding. Otherwise, cells will be crushed to one corner of each well, and the plate will not be suitable for high-content imaging.</li>
		<li>Incubate the 384-well plate at 37 &#176;C with 5% CO2 for 3 h for the cells to attach to the bottom of the plate.</li>
	</ol>
	</li>
</ol>
3. Treating the Cells with Compounds
<ol>
	<li>Generate a plate map with treatment conditions as shown in Figure 2.</li>
	<li>Prepare 300 &#181;L of 5x working solutions of up to 15 compounds in a 96-well plate.
	<ol>
		<li>Dilute cps 1 to 15 with the assay medium to five times of their final concentrations in wells A1 to G2 of the 96-well plate (Figure 2A). Add 300 &#181;L of assay medium in well H2. 
		NOTE: The final concentration of each compound is used at the most effective concentration for rescuing the transport of the P23H rhodopsin by the HTS12,13.</li>
		<li>Add 100 &#181;L per well of 2% dimethyl sulfoxide (DMSO) and 25 &#181;M 9-cis-retinal that are diluted in the assay medium to columns 11 and 12, respectively. Add 9-cis-retinal in dim light.</li>
	</ol>
	</li>
	<li>Adding 10 &#181;L per well of 5x working solutions to the 384-well plate cultured with the cells (Figure 2B).
	<ol>
		<li>Use an electronic multi-channel pipette to add compounds 1, 3, 5, 7, 9, 11, 13, and 15 to rows A, C, E, G, I, K, M, and O from columns 1 to 21 (Figure 2). Add compounds 2, 4, 6, 8, 10, 12, 14 and M to rows B, D, F, H, J, L, N, and P from columns 1 to 21.</li>
		<li>Add 10 &#181;L per well of 2% DMSO to columns 22 and 24. Add 10 &#181;L per well of 25 &#181;M 9-cis-retinal to column 23. 
		NOTE: DMSO is used as a vehicle control because all the compounds are initially dissolved in DMSO as stocks. Column 22 containing DMSO treated cells expressing the P23H rhodopsin is used as the 0% control. 9-Cis-retinal treated cells expressing the P23H rhodopsin is used as the 100% control.</li>
	</ol>
	</li>
	<li>Cover the 384-well plate with aluminum foil and incubate the plate at 37 &#176;C for 24 h.</li>
</ol>
4. Immunostaining without Membrane Permeabilization to Stain Rhodopsin Protein on the Cell Surface.
NOTE: Avoid any detergent in the entire immunostaining process to keep cell membrane intact.
<ol>
	<li>Prepare 4% paraformaldehyde (PFA) by diluting the 16% PFA with PBS in a 1:4 volume ratio in a chemical fume hood. Transfer the 4% PFA in a reagent reservoir.</li>
	<li>Take out the 384-well plate in a dark room with dim red light. Use an 8-channel aspirator connected to a vacuum collection bottle to gently aspirate the medium. Use an electronic multichannel pipette to add 20 &#181;L per well of freshly prepared 4% PFA to the entire 384-well plate and incubate for 20 min at room temperature. To avoid cell detachment, always point the tips of the aspirator to the same side of each well during aspirations. Do not touch the middle bottom area of each well. When taking images, select fields to avoid the region touched by the aspirator. For incubations longer than 10 min, cover the 384-well plate with its lid to avoid evaporation. 
	NOTE: Cells are fixed in a dark room to avoid photobleaching of the regenerated isorhodopsin that will affect the result of the 100% control. After fixation, the 384-well plate can be taken out under normal light.</li>
	<li>Use an 8-channel aspirator to aspirate the PFA in each well and use an electronic multi-channel pipette to add 50 &#956;L per well of PBS. Repeat two more times to perform three washes with PBS. 
	CAUTION: The waste liquid containing PFA are collected in a capped bottle and is to be disposed of as a hazardous chemical waste after the experiment.</li>
	<li>Block the cells by adding 20 &#181;L per well of 5% goat serum to the entire 384-well plate and incubate at room temperature for 30 min.</li>
	<li>Aspirate the 5% goat serum and add 15 &#181;L per well of 20 &#181;g mL-1 B6-30 anti-rhodopsin antibody in 1% goat serum to rows A to O. Add 15 &#181;L per well of 1% goat serum to row P for the secondary antibody-only control group.</li>
	<li>Incubate the 384-well plate at room temperature for 90 min or at 4 &#176;C overnight. Cover the 384-well plate with aluminum foil to avoid photobleaching of the fluorophores.</li>
	<li>Wash the plate 3 times with 50 &#181;L per well of PBS.</li>
	<li>Aspirate PBS and add 15 &#181;L per well of 5 &#181;g mL-1 Cy3-conjugated goat anti-mouse IgG antibody. Cover the 384-well plate with aluminum foil and incubate at room temperature for 1 h or at 4 &#176;C overnight.</li>
	<li>Wash the plate 3 times with 50 &#181;L per well of PBS. Add 50 &#181;L per well of PBS containing 1 &#181;g mL-1 Hoechst 33342 to stain nuclei at room temperature for 15 min.</li>
	<li>Seal the 384-well plate with a transparent film before imaging. Cover the 384-well plate with aluminum foil and store at 4 &#176;C for up to a week if images are taken immediately.</li>
</ol>
5. Imaging.
NOTE: This high-content imaging procedure is adapted to the imaging system listed in the Table of Material. Procedures can be different if using other high-content imaging systems.
<ol>
	<li>Remove the lid and put the 384-well plate into the high-content imager with A1 positioned on the top left corner of the plate.</li>
	<li>Open the image acquisition software to set up parameters for image acquisition.
	<ol>
		<li>Open the Plate Acquisition Setup window and create new setting or load an existing setup file.</li>
		<li>Select the 20x objective and set up pixel binning as 2 so the calibrated pixel size is 0.80 x 0.80 &#181;m. Set the scan lines as 2,000 and the image size (W x H) as 1,000 x 1,000 pixels (800.00 x 800.00 &#181;m) per site.</li>
		<li>Select the plate type to be imaged. Use the information provided by the manufacturer of the 384-well plate to fill in the plate dimensions.</li>
		<li>Select the wells to be imaged for the 384-well plate.</li>
		<li>Select four sites to be imaged per well. Avoid the side of the well touched by the aspiration tips.</li>
		<li>Select excitation lasers as 405, 488 and 561 nm. Select emission filters for DAPI, FITC and Texas Red channels. Optimize the laser power and gains of each channel to ensure the brightness of images taken from the positive control wells are less than the saturation threshold.</li>
		<li>Select well-to-well focus for autofocus. Select the first well acquired as the initial well for finding samples. Set site autofocus to all sites.</li>
		<li>Select four averages per line for each channel. Optimize the Z-offset value for each channel.</li>
		<li>Test 2-3 wells at the diagonal corners of the 384 well plate to make sure the images are on focus for all tested wells, images from all sites per well have cells with more than 40% confluence, and fluorescence intensities of all channels are about half saturated in the 100% control wells.</li>
		<li>Save the image acquisition method.</li>
	</ol>
	</li>
	<li>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. Take the 384-well plate out and store at 4 &#176;C for future use. 
	NOTE: Depending on the number of sites selected per well and the channels taken, it takes 40 min to 3 h to finish imaging one 384-well plate.</li>
</ol>
6. Image Analysis.
<ol>
	<li>Pull out the image data using a high-content image analysis software. Select one of the 100% control wells (column 23) to set up parameters.</li>
	<li>Select the Multi-Wavelength Cell Scoring as the analysis method and start configuring settings.
	<ol>
		<li>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.</li>
		<li>Define the shape of cell in the FITC channel where rhodopsin-Venus is imaged.</li>
		<li>Define the rhodopsin cell surface stain areas in the Texas Red channel.</li>
		<li>Test the current algorithm in 5 wells to determine if the settings are optimized. Save the settings and close the Configuration window.</li>
	</ol>
	</li>
	<li>Run all the wells with the optimized analysis method.</li>
	<li>Export Object Number as intact cell number. Export the Average Intensity of the FITC channel as Rhodopsin-Venus Intensity (Rhodopsin-Venus INT). Export the Average Intensity of the Texas Red channel as rhodopsin intensity on the cell surface (Rhodopsin INT on the cell surface).</li>
	<li>Open the exported data in a spreadsheet software. Divide the rhodopsin intensity on the cell surface by the rhodopsin-Venus INT as MEM-to-total ratio. Calculate the Z&#8217;-factors for each parameter to evaluate the assay quality. Z&#8242;=1&#8722;3X(STD100% control+STD0% control)/|Mean100% control&#8722;Mean0% control|. 
	NOTE: A Z&#8217;-factor higher than 0 indicates a moderate assay sufficient for a high-content screen, and a Z&#8217; factor between 0.5 and 1 suggests an outstanding assay required for a high-throughput screen20,21.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

Here, we showed a high-content imaging assay used for characterizing hits identified from a HTS. The only automation involved in these protocols is the high-content imager. The immunostaining and fluorescence imaging of rhodopsin have been used commonly to characterize the localization of rhodopsin5,14,15,16. However, the quantification of images taken by the traditional imaging methods is limi...

Protein misfolding is involved in muscular dystrophy, neural degenerations, as well as blinding diseases, including retinitis pigmentosa (RP)1. RP is an inherited and progressive retinal degeneration associated with mutations in over 60 genes affecting the function and homeostasis of rod photoreceptors or the retinal pigmented epitheliums (RPEs)2,3. No effective treatment is currently available for RP. Rhodopsin mutations account for about 25-30% of autosomal dominant (ad) RP cases. Among the more than 150 rhodopsin mutations4 (Human Gene Mutation Database, http:/www.hgmd.cf.ac.uk/), the Class II mutations cause the structural instability of the rhodopsin protein that contributes to the rod photoreceptor death and vision loss5,6,7,8. The P23H is the most frequent rhodopsin mutation in North America, which is also a typical example of the Class II rhodopsin mutations9,10. Due to its inherent structural instability, the misfolded rhodopsin is accumulated in the endoplasmic reticulum (ER) in mammalian cells, whereas the wild type rhodopsin is located on the plasma membrane5. The misfolded rhodopsin P23H mutant exhibits dominant negative cytotoxicity that is not due to haploinsufficiency, but is related to the activation of ER associated protein degradation pathway and the interrupted rod outer segment organization. To alleviate rod photoreceptor cell stress, one strategy is to stabilize the native folding of the mutant rhodopsin using a pharmacological chaperone.
To achieve this goal, we performed a cell-based high-throughput screen (HTSs)11,12,13 using a &#946;-galactosidase fragment complementation assay to quantify the P23H rhodopsin mutant transported on the plasma membrane. The robust and simple protocol of this HTS assay enabled us to explore the activities of about 79,000 small molecules for each screen. However, because this HTS assay reads luminescence signals, false positives including the &#946;-gal inhibitors, colored or cytotoxic compounds are included in the hit list waiting to be identified by a secondary assay.
The traditional immunostaining and fluorescence imaging methods have been used for years to study the rhodopsin transport in mammalian cells5,14,15,16. However, these conventional methods cannot be used to quantify pharmacological effects of more than 10 compounds towards rhodopsin transport because a reliable imaging analysis requires a large number of images taken under a highly consistent condition, which is not amendable by the conventional imaging methods. Here, we developed an immunostaining based high-content imaging protocol as a secondary assay to quantify the cell surface transport of misfolded rhodopsin mutants11,13,17. To label rhodopsin on the plasma membrane, we skipped the step of cell membrane permeabilization and immunostained the rhodopsin mutants by a monoclonal (B6-30) anti-rhodopsin recognizing the N-terminal epitope of rhodopsin at the extracellular side of the cell membrane18. To visualize the mutant rhodopsin in the whole cell, we fused rhodopsin with the Venus fluorescence protein. By the quantification of the fluorescence intensities in different fluorescence channels, we are able to obtain multiple parameters from one single experiment including the total rhodopsin intensity in the whole cell, on the cell surface, and the ratio of rhodopsin fluorescence on the cell surface to that in the whole cell. Applying this method to stable cells expressing a total of six misfolded rhodopsin mutants, we can generate a pharmacological profile of multiple small molecule chaperones towards these mutants. In this protocol, all cells are immunostained in a 384-well plate and imaged using an automated imaging system under a highly consistent imaging condition. An image analysis is performed to each well, containing images of more than 600 cells to reduce variation due to the heterogeneity of the cells with varying cell shape and protein expression level. The workflow of this protocol is summarized in Figure 1. The advantage of this method is that we obtain high-resolution images as well as multi-parameter quantifications from the image-based analysis. In general, this protocol can be modified and applied to quantify the transport of any misfolded membrane protein of interest.

<table>
	<tbody>
		<tr>
			<td>U2OS (rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (T4R-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P23H-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P53R-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (C110Y-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (D190N-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P267L-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Genesee Scientific</td>
			<td>25-500</td>
			<td>With L-Glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>InvivoGen</td>
			<td>ant-mpt</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>100X concentrated antibiotic solutions to prevent bacteria contamination of cell cultures</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>0.25%, 1mM EDTA in HBSS without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4707-50ML</td>
			<td>Mol wt 70,000-150,000, 0.01%, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>CellCarrier-384 Ultra Microplates</td>
			<td>PerkinElmer</td>
			<td>6057300</td>
			<td>384-well tissue culutre-treated microplates with black well walls and an optically -clear cyclic olefin bottom for imaging cells in high content analysis</td>
		</tr>
		<tr>
			<td>Sterile 96-well plate</td>
			<td>Eppendorf</td>
			<td>30730119</td>
			<td>Tissue culture treated with lid flat bottom, sterile, free of detectable pyrogens, Rnase, DNase and DNA. Non-cytotoxic</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Sailine (PBS)</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td>10 x PBS Buffer, pH 7.4</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D4540</td>
			<td>&#62;99.5%, cell culture tested</td>
		</tr>
		<tr>
			<td>9-cis-retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R5754</td>
			<td></td>
		</tr>
		<tr>
			<td>Compounds tested</td>
			<td>Selleckchem/Life Chemicals/Custom synthesized</td>
			<td>NA</td>
			<td>Compounds were purchased from different vendors or custom synthesized</td>
		</tr>
		<tr>
			<td>B6-30 anti-rhodopsin antibody</td>
			<td>Novus</td>
			<td>NBP2-25160</td>
			<td>Gift from Dr. Krzysztof Palczewski</td>
		</tr>
		<tr>
			<td>Cy3-conjugated goat anti-mouse secondary antibody</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>115-165-146</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Methanol-free</td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>50062Z</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydroch</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Nuclear staining solution</td>
		</tr>
		<tr>
			<td>High-content imager</td>
			<td>Molecular Devices</td>
			<td>ImageXpress</td>
			<td>ImageXpress&#174; Micro Confocal High-Content Imaging System</td>
		</tr>
		<tr>
			<td>MetaXpress high-content image acquisition and analysis software</td>
			<td>Molecular Devices</td>
			<td>MetaXpress</td>
			<td>High-content image acquisition and analysis software</td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5-10 &#181;L)</td>
			<td>Rainin</td>
			<td>17013802</td>
			<td>Manual 8-channel pipette, 0.5-10 &#181;L</td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5-10c</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td>Manual 8-channel pipette, 20-200 &#181;L</td>
		</tr>
		<tr>
			<td>Electronic multichannel pipette (10-200 &#956;L)</td>
			<td>Thermo Scientific</td>
			<td>14-3879-56BT</td>
			<td>Electronic multichanenel pipette for 96- and 384-well microplate pipetting tasks</td>
		</tr>
		<tr>
			<td>50ml Reagent Reservoir</td>
			<td>Genesee Scientific</td>
			<td>28-125</td>
			<td>Reagent reservior for multichannel pippte dispensing</td>
		</tr>
		<tr>
			<td>8-Channel aspirator</td>
			<td>ABC Scientific</td>
			<td>EV503</td>
			<td>8-Channel stainless steel adaptor for aspirating liquids from 96- or 384-well plates</td>
		</tr>
		<tr>
			<td>Excel spreadsheet software</td>
			<td>Microsoft</td>
			<td>Excel2016</td>
			<td>The spreadsheet software for data analysis and heatmap generation</td>
		</tr>
		<tr>
			<td>Origin2018 scientific data analysis and graphing software</td>
			<td>OriginLab</td>
			<td>Origin2018</td>
			<td>The data analysis software for generating the dose response curves</td>
		</tr>
	</tbody>
</table>

a rhodopsin transport assay by high-content imaging analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

We characterized the rhodopsin transport with three parameters: the rhodopsin-Venus intensity in the whole cell (Rhodopsin-Venus INT), the immunostaining intensity of rhodopsin on the plasma membrane (Rhodopsin INT on the cell surface), and the ratio of rhodopsin stain on the cell surface to rhodopsin-Venus intensity in the whole cell (MEM-Total Ratio). A representative result of the rhodopsin transport assay is shown in Figure 3 and Figu...

Watch this Scientific Journal Video about A Rhodopsin Transport Assay by High-Content Imaging Analysis at JoVE.com

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

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6.4K Views.  University of Pittsburgh. Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Video: A Rhodopsin Transport Assay by High-Content Imaging Analysis

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

The Effect of Charging and Discharging Lithium Iron Phosphate-graphite Cells at Different Temperatures on Degradation

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. The degradation of the cells is assessed by using 10 charging and discharging temperature permutations ranging from -20 &#176;C to 30 &#176;C. This allows an analysis of the effect of charge and discharge temperatures on aging, and their associations. A total of 100 charge/discharge cycles were carried out. Every 25 cycles a reference cycle was performed to assess the reversible and irreversible capacity degradation. A multi-factor analysis of variance was used, and the experimental results were fitted showing: i) a quadratic relationship between the rate of degradation and the temperature of charge, ii) a linear relationship with the temperature of discharge, and iii) a correlation between the temperature of charge and discharge. It was found that the temperature combination for charging at +30 &#176;C and discharging at -5 &#176;C led to the highest rate of degradation. On the other hand, the cycling in a temperature range from -20 &#176;C to 15 &#176;C (with various combinations of temperatures of charge and discharge), led to a much lower degradation. Additionally, when the temperature of charge is 15 &#176;C, it was found that the degradation rate is nondependent on the temperature of discharge.

This article describes the effect of dissimilar charging/discharging temperatures on the degradation of lithium iron phosphate-graphite pouch cells, aiming at simulating close to real case scenarios. In total, 10 temperature combinations are investigated in the range -20 to 30 &#176;C in order to analyze the impact of temperature on degradation.

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.