Name: two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis
Uploaded: 2021-12-07T13:00:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

This work was supported by NIH Grant EY12155, EY027193, and EY027387 to JC. We are thankful to Dr. Spyridon Michalakis (Caltech, Pasadena, USA) and Natalie Chen (USC, Los Angeles, USA) for proofreading the manuscript. We would also like to thank Dr. Seth Ruffins (USC, Los Angeles, USA) and Dr. Janos Peti-Peterdi (USC, Los Angeles, USA) for providing the necessary equipment to collect the author provided footage. Material from: Kasey Rose et al, Separation of photoreceptor cell compartments in mouse retina for protein analysis, Molecular Neurodegeneration, published [2017], [Springer Nature].

All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Southern California (USC).
1. Live cell retinal peeling method
<ol>
	<li>Preparation of Ames&#39; buffers, peeling papers, and dissection dish
	<ol>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper (Grade 413) into rectangles measuring approximately 5 mm x 2.5 mm. Store the cuttings for future use.</li>
		<li>Prepare 1 L of Ames&#39; HEPES buffer by combining one bottle of Ames&#39; Medium, 2.38 g HEPES, and 0.877 g of NaCl. Dissolve the reagents in a sterile glass bottle with distilled ultrapure water and adjust the osmolarity and pH to 280 mOsm and 7.4, respectively. Filter to sterilize (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C.</li>
		<li>Prepare 1 L of Ames&#39; bicarbonate buffer by combining one bottle of Ames&#39; Medium and 1.9 g of NaHCO3. Dissolve the reagents in a sterile glass bottle with distilled ultrapure water and adjust the osmolarity and pH to 280 mOsm and 7.4, respectively. Filter to sterilize (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C. 
		NOTE: Ames&#39; HEPES and Ames&#39; bicarbonate buffers can be prepared in advance and stored for 1 week at 4 &#176;C.</li>
		<li>At the bottom of a 35 x 10 mm Petri dish, create a lattice or checkerboard pattern with a scalpel (blade No. 11, 40 mm). 
		NOTE: The textured bottom of the Petri dish holds the isolated, free-floating eye in place during the retinal dissection.</li>
	</ol>
	</li>
	<li>Dissection of the retina 
	NOTE: For this procedure, the buffer should be brought to and kept at room temperature (RT). Refresh the Ames&#39; bicarbonate buffer every 15 min with fresh carbogenated Ames&#39; bicarbonate buffer during the dissection. This maintains the necessary physiologic pH.
	<ol>
		<li>Around 15-20 min before the dissection, oxygenate the Ames&#39; HEPES buffer in a 100 mL laboratory or media bottle equipped with a tubing cap adapter and bubble the Ames&#39; Bicarbonate buffer with 95% O2 and 5% CO2 in a tissue incubation chamber and in a 100 mL laboratory or media bottle equipped with a tubing cap adapter.</li>
		<li>Euthanize an equal number of both genders of C57BL/6J mice (2-3 months old) either by isoflurane inhalation or carbon dioxide (CO2) followed by cervical dislocation. Enucleate the eyes with curved scissors and place the eyes into the textured 35 x 10 mm Petri dish filled with bubbled Ames&#39; bicarbonate buffer.</li>
		<li>Create an eye cup (Figure 1A) by puncturing a hole at the cornea-limbus junction with an 18 G needle. Cut along the perimeter of this junction using micro-dissection iris scissors to remove the cornea of each eye. Separate the lens and vitreous humor from each eye using tweezers and discard.</li>
		<li>Isolate the retina from the eye cup by carefully peeling the retinal pigmented epithelium (RPE)-sclera-choroid complex away from the neural retina. Discard the RPE-sclera-choroid complex. Ensure that the retina is not damaged during the dissection by handling only the edges.</li>
		<li>Transfer isolated retinae using a wide bore transfer pipette into the lid of a 60 x 15 mm dish filled with bubbled Ames&#39; bicarbonate buffer.</li>
		<li>Orient the hemisphere-shaped retina concave up (the photoreceptors are facing down toward the bottom of the dish) and bisect each retina (through the optic nerve) using iris scissors or a scalpel (blade No. 10, 40 mm). Trim the curved edges of each half retina to make two rectangles (Figure 1A). 
		NOTE: Minimizing the curvature of each halved retina allows for the retina to flatten better in subsequent peeling steps and produces an accurate peel of the rod outer segment (ROS) layer.</li>
		<li>Store the halved retinae in the tissue incubation chamber that is continuously bubbled with 95% O2 and 5% CO2. 
		NOTE: Isolated retinae can be kept in the carbogenated Ames&#39; bicarbonate buffer for ~24 h.</li>
	</ol>
	</li>
	<li>Rod outer segment (ROS) collection by filter paper peeling 
	NOTE: Refresh the RT oxygenated Ames&#39; HEPES buffer every 15-20 min during the peeling process to maintain the necessary physiologic pH.
	<ol>
		<li>Transfer one retinal rectangle from the tissue chamber with a wide bore transfer pipette and a 5 mm x 2.5 mm piece of filter paper into a 35 x 10 mm Petri dish filled with Ames&#39; HEPES buffer bubbled with 100% O2.</li>
		<li>Orient the partitioned retina concave up and ensure the photoreceptors are facing down toward the bottom of the dish. Lightly grasp the sides of the halved retina with tweezers and move it on top of the filter paper. Once the retina is centered on the filter paper, move the filter paper upward so the halved retina touches the filter paper to create the ROS-to-filter paper adhesion (Figure 1B). 
		NOTE: Make sure that the photoreceptor layer makes direct contact with the filter paper.</li>
		<li>Carefully lift the filter paper with the attached retina out of the Ames&#39; HEPES buffer. Place the bottom side of the filter paper (the side without the retina) on a paper towel and dab 2-3 times to dry (Figure 1B).</li>
		<li>Add a drop of Ames&#39; HEPES onto the side of the filter paper with the retina. Place the filter paper on the paper towel again to dry, as just described. Repeat this process two more times.</li>
		<li>Place the filter paper with the retina back into the Petri dish. Using tweezers, gently push all edges of the halved retina up and away from the filter paper on all sides.</li>
		<li>Delicately peel the retina away from the filter paper. Touch only the extreme perimeter of the retina during the peeling process to preserve the retina&#39;s structural integrity.</li>
		<li>Lift the filter paper from the Petri dish and remove excess liquid on a paper towel. Make sure the side that was in contact with the retina does not contact the paper towel.</li>
		<li>Place the filter paper with the partial ROS layer into a labeled tube on ice (Figure 1B). Keep the peeled retina submerged in Ames&#39; HEPES buffer.</li>
		<li>Repeat the peeling process for this halved retina approximately 7-8 times to remove the entire ROS layer. After each peel, place the filter paper into the same tube, which will contain the isolated ROS from one halved retina. 
		&#8203;NOTE: Take care to not tear the halved retina when peeling it away from the filter paper, as the retina becomes thinner during this process.</li>
		<li>Place the tube containing all filter paper peels of the isolated ROS on ice for immediate use or freeze directly on dry ice and store at -80 &#176;C.</li>
		<li>Use tweezers to transfer the leftover peeled retina (lacking ROS) into an appropriately labeled tube. Place the tube on ice for immediate use or freeze with dry ice and store at -80 &#176;C.</li>
		<li>Repeat the peeling process for each halved retina and accurately label each tube.</li>
	</ol>
	</li>
</ol>
2. Lyophilized retina peeling method
<ol>
	<li>Buffers, peeling medium, dissection dish preparation, and lyophilizer set-up
	<ol>
		<li>In a 1 L storage bottle, prepare the Ringer&#39;s solution by adding 500 mL of distilled ultrapure water. Dissolve the reagents to reach a final concentration of 130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, and 0.02 mM EDTA. Adjust the pH to 7.4 with NaOH, add ultrapure water to bring the final volume to 1 L, and adjust osmolarity to 313 mOsm.</li>
		<li>Prior to the use, filter the Ringer&#39;s solution with a sterile filter (pore size 0.2 &#181;m), seal the lid with paraffin film, and store at 4 &#176;C.</li>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper into rectangles measuring approximately 5 mm x 2.5 mm. Use a pencil to write on one side of the filter paper to create a unique label.</li>
		<li>Create a lattice or checkerboard pattern in the bottom of a 35 x 10 mm Petri dish using a scalpel (blade No. 11, 40 mm).</li>
		<li>Turn on the lyophilizer according to the manufacturer&#39;s specifications.</li>
		<li>Acquire liquid nitrogen in a Dewar flask or similar insulating vacuum flask.</li>
	</ol>
	</li>
	<li>Retinal dissection
	<ol>
		<li>Complete the retinal dissection as described in section 1 of the protocol (Figure 1A). Use cold Ringer&#39;s solution instead of Ames&#39; buffer.</li>
		<li>Post dissection, store the halved, rectangular retinae in a 35 x 10 mm Petri dish filled with cold Ringer&#39;s solution and place on ice.</li>
	</ol>
	</li>
	<li>Retinal sample preparation for lyophilization
	<ol>
		<li>Place a 5 x 2.5 mm piece of filter paper and transfer a halved retina into the texturized 35 x 10 mm Petri dish filled with cold Ringer&#39;s buffer. Use a wide bore transfer pipette to move each retina between dishes.</li>
		<li>Use tweezers to carefully flip the retina so it is oriented concave up (the photoreceptors are pointing upward) and move it so it rests on top of the filter paper (Figure 2A). 
		NOTE: Make sure the photoreceptors do not contact the filter paper (the retinal ganglion cell layer should be in direct contact with the filter paper) and the unique label is visible. To easily and quickly identify the retinal isolate, it is recommended that the unique label should be located on the underside of the filter paper.</li>
		<li>Lift the filter paper by the edges upward so the halved piece of retina touches the filter paper and continue to lift the filter paper out of the Ringer&#39;s solution.</li>
		<li>Place the bottom side of the filter paper (the side without the retina on it) on a paper towel and carefully dab 2-3 times to remove excess liquid (Figure 2A).</li>
		<li>Pick up the filter paper with tweezers and add a drop of cold Ringer&#39;s onto the side of the filter paper with the retina. Place the filter paper on the paper towel again to remove excess liquid. Repeat the process two more times. 
		NOTE: These alternating wetting and drying steps ensure that the tissue is firmly attached to the filter paper.</li>
		<li>Store each adhered retina/filter paper sample in a Petri dish filled with cold Ringer&#39;s until ready to freeze all samples. Repeat this process for all halved retinae.</li>
		<li>Obtain a clean and dry 35 x 10 mm Petri dish (bottom only) and a piece of aluminum foil cut into a 3.5 x 3.5 inch square.</li>
		<li>Lift each sample out of the cold Ringer&#39;s buffer with tweezers. Ensure that the tweezers contact only the filter paper edges, avoiding the halved retina.</li>
		<li>Place the bottom side of each filter paper (the side without the retina on it) on a paper towel and remove excess liquid.</li>
		<li>Add a drop of cold 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) onto the side of the filter paper where the retina is adhered. Dab the filter paper on the paper towel to remove excess liquid. 
		NOTE: Make sure that the moisture from the filter paper is sufficiently removed before freezing.</li>
		<li>Place all filter paper pieces into the 35 x 10 mm Petri dish. Use a lint free tissue paper to wick away any excess liquid surrounding the filter paper. 
		NOTE: Do not overcrowd the Petri dish with samples. If more than four murine eyes are used, consider using a larger Petri dish or multiple smaller Petri dishes to hold the retina/filter paper samples.</li>
		<li>Tightly wrap the entire dish with aluminum foil. Ensure that the edges of the aluminum foil are secured and smoothly pressed into the bottom of the Petri dish. Puncture a handful of 0.1-0.2 mm holes into the aluminum foil lid (Figure 2A).</li>
		<li>Using metal tongs, gradually lower the aluminum foil covered Petri dish into the liquid nitrogen. Keep the samples in the liquid nitrogen (&#60;10 min) until ready to lyophilize.</li>
	</ol>
	</li>
	<li>Lyophilization
	<ol>
		<li>Place the aluminum foil covered Petri dish into a freeze-drying flask and attach it to the lyophilizer machine following the manufacturer&#39;s protocol. Lyophilize the retinae for 30 min.</li>
		<li>Detach the flask from the lyophilizer and shut off the machine according to the manufacturer.</li>
		<li>Remove the aluminum foil and either work with the freeze-dried samples the same day or store them in properly labeled 1 mL microcentrifuge tubes. Place these tubes in a container (such as a 50 mL conical tube) filled with an anhydrous desiccant and store the samples at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Rod outer and inner segment collection by adhesive tape peeling.
	<ol>
		<li>Cut strips of clear adhesive tape (1-2 cm) and store strips on the edge of the tape dispenser/lab bench/etc. for quick use.</li>
		<li>Move one retinal sample to the lid of a plastic 60 x 15 mm Petri dish for peeling. Ensure to only grab the outermost edges of the filter paper and avoid touching the retina.</li>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut a small rectangular piece of tape to the approximate size of the tissue (1.5 x 2.5 mm).</li>
		<li>Carefully lay a small piece of tape on top of the lyophilized retina (Figure 2B) and apply slight pressure with the tweezers to make sure it bonds with the photoreceptor layer (the orange/pink-tinted top layer).</li>
		<li>Slowly peel the tape away. Both the rod outer segment (ROS) and rod inner segment (RIS) will be adhered to the tape (Figure 2B).</li>
		<li>Ensure that there is a thin white film at the fractured surface. This is RIS.To separate the RIS, place another piece of tape and push the tape down on the fractured surface (Figure 2B). 
		NOTE: It may take more than one piece of tape to remove the entire thin white layer.</li>
		<li>Place the piece of tape with only the orange/pink layer into a microcentrifuge tube labeled +ROS.</li>
		<li>Place the tape or pieces of tape with the thin white layer into a microcentrifuge tube labeled +RIS. 
		NOTE: Separating the lyophilized retina with tape takes practice. The amount of pressure applied to the tape will affect how the lyophilized sample will fraction. If too much pressure is placed on the tape over the sample, the whole lyophilized retina will peel off the filter paper and will stick to the tape. If too little pressure is added to the tape, the orange/pink top layer (+ROS) will not stick to the tape.</li>
		<li>Collect the leftover retinal tissue (bereft of ROS and RIS layers, -OIS) located on the filter paper by peeling off the thick, white layer from the filter paper using tape. Place the isolate into a tube labeled -OIS.</li>
		<li>Repeat these steps for each halved retinal sample.</li>
		<li>Store the isolated layers from the lyophilized retinae either at room temperature if performing protein quantification, gel electrophoresis, and protein immunoblots that same day, or store in a container (such as 50 mL conical tube) filled with anhydrous desiccant at -80 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
3. Western blot sample preparation for peeling isolations
<ol>
	<li>Prepare RIPA lysis buffer
	<ol>
		<li>In a 100 mL storage bottle, add reagents to obtain a final concentration of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, and 1 mM EDTA. Add 100 mL of deionized ultrapure water and mix thoroughly. 
		NOTE: RIPA lysis buffer can be stored at 2-8 &#176;C for 2-3 weeks. For longer storage, aliquot and store in the -20 &#176;C freezer.</li>
		<li>On the day of the experiment, add a protease inhibitor cocktail (0.1 M PMSF, 1:1000 Aprotinin, and 1:1000 Leupeptin) to the RIPA lysis buffer and keep on ice.</li>
	</ol>
	</li>
	<li>Filter paper peeling lysate preparation for western blot
	<ol>
		<li>Ensure that tubes containing the filter paper peels and leftover peeled retinae are maintained on ice (for the experiment on the same day) or removed from -80 &#176;C freezer storage and placed on ice.</li>
		<li>Remove any excess liquid from the bottom of the microcentrifuge tubes containing the filter paper peels. Quickly spin in a mini centrifuge for 2-4 s and discard any remaining liquid.</li>
		<li>Pipette 45-55 &#181;L of cold RIPA buffer into tubes containing the filter paper isolate.</li>
		<li>Homogenize each sample with a clean, autoclaved pestle mixer for 1 min. Make sure the filter paper stays in contact with the pestle mixer. Keep the filter paper on the side of each tube rather than at the bottom.</li>
		<li>With tweezers, move the filter papers to the side of each tube and spin in the mini centrifuge for 2-4 s. This will remove the RIPA buffer from the filter paper.</li>
		<li>Remove dried filter paper and repeat for all filter papers within each tube if necessary.</li>
		<li>Transfer the homogenate to a new tube and label it appropriately. 
		NOTE: This is the most time-consuming step, and if not completed properly, much of the sample will end up being absorbed by the filter paper.</li>
		<li>Pipette 60-80 &#181;L of cold RIPA buffer into the individual tubes with the leftover peeled retinae.</li>
		<li>Homogenize each sample with a clean, autoclaved pestle mixer for 1 min.</li>
	</ol>
	</li>
	<li>Tape peeling Lysate preparation for western blot
	<ol>
		<li>Place the RT lyophilized samples or the -80 &#176;C samples on ice.</li>
		<li>Pipette 50-70 &#181;L of cold RIPA buffer into each tube containing +ROS and +RIS tape peels.</li>
		<li>Pipette 60-80 &#181;L of cold lysis buffer into the -OIS tubes containing the remaining lyophilized retina, with the ROS and RIS removed.</li>
		<li>Homogenize each sample with a clean pestle mixer for 1 min. Ensure that the tape in the individual tubes stays in contact with the pestle mixer and lysis buffer.</li>
		<li>With sterilized tweezers, move the tape to the side of the tubes and spin with a mini centrifuge for 2-4 s. This will remove the liquid from the tape. Remove the dried tape from the tubes. 
		NOTE: At this point, you may combine two half retinal isolates from the same retina for either the &#39;filter paper peeling&#39; or &#39;tape peeling&#39; method to ensure you have enough volume to perform a bicinchoninic acid assay (BCA).</li>
	</ol>
	</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+retinal+outer+segment+fragments.">The isolation of retinal outer segment fragments.</a> The Journal of Cell Biology. 27 (3), 459-473 (1965).</li><li>Tsang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+target+enzyme+in+deactivation+of+photoreceptor+G+protein+in+vivo.">Role for the target enzyme in deactivation of photoreceptor G protein in vivo.</a> Science. 282 (5386), 117-121 (1998).</li><li>Walston, S. T., Chow, R. H., Weiland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recordings+of+retinal+bipolar+cells+in+response+to+extracellular+electrical+stimulation+in+wholemount+mouse+retina.">Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2015, 3363-3366 (2015).</li><li>Guido, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+obtain+retinal+cell+preparations+highly+enriched+in+specific+cell+types.+Suitability+for+lipid+metabolism+studies.">A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.</a> Brain Research. Brain Research Protocols. 4 (2), 147-155 (1999).</li><li>Hayashi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+by+cyclin-dependent+protein+kinase+5+of+the+regulatory+subunit+of+retinal+cGMP+phosphodiesterase.+II.+Its+role+in+the+turnoff+of+phosphodiesterase+in+vivo.">Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.</a> The Journal of Biological Chemistry. 275 (42), 32958-32965 (2000).</li><li>Wolbring, G., Cook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+and+characterization+of+protein+kinase+C+from+bovine+retinal+rod+outer+segments.">Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments.</a> European Journal of Biochemistry. 201 (3), 601-606 (1991).</li><li>Williams, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+kinase+C+in+photoreceptor+outer+segments.">Characterization of protein kinase C in photoreceptor outer segments.</a> Journal of Neurochemistry. 69 (4), 1693-1702 (1997).</li><li>Watson, A. J., Aragay, A. M., Slepak, V. Z., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+form+of+the+G+protein+beta+subunit+Gbeta5+is+specifically+expressed+in+the+vertebrate+retina.">A novel form of the G protein beta subunit Gbeta5 is specifically expressed in the vertebrate retina.</a> The Journal of Biological Chemistry. 271 (45), 28154-28160 (1996).</li><li>Makino, E. R., Handy, J. W., Li, T., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GTPase+activating+factor+for+transducin+in+rod+photoreceptors+is+the+complex+between+RGS9+and+type+5+G+protein+beta+subunit.">The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (5), 1947-1952 (1999).</li><li>Chen, J., Shi, G., Concepcion, F. A., Xie, G., Oprian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+rhodopsin/arrestin+complex+leads+to+retinal+degeneration+in+a+transgenic+mouse+model+of+autosomal+dominant+retinitis+pigmentosa.">Stable rhodopsin/arrestin complex leads to retinal degeneration in a transgenic mouse model of autosomal dominant retinitis pigmentosa.</a> The Journal of Neuroscience. 26 (46), 11929-11937 (2006).</li><li>Chen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+photoresponses+and+light-induced+apoptosis+in+rods+lacking+rhodopsin+kinase.">Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3718-3722 (1999).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mistrafficking+of+prenylated+proteins+causes+retinitis+pigmentosa+2.">Mistrafficking of prenylated proteins causes retinitis pigmentosa 2.</a> FASEB Journal. 29 (3), 932-942 (2015).</li><li>Weiss, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+differences+in+expression+of+G-protein-coupled+receptor+kinase+(GRK)+7+and+GRK1+in+mammalian+cone+photoreceptor+cells:+implications+for+cone+cell+phototransduction.">Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.</a> The Journal of Neuroscience. 21 (23), 9175-9184 (2001).</li><li>Zhao, X., Huang, J., Khani, S. C., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forms+of+human+rhodopsin+kinase+(GRK1).">Molecular forms of human rhodopsin kinase (GRK1).</a> The Journal of Biological Chemistry. 273 (9), 5124-5131 (1998).</li><li>Newton, A. C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+protein+kinase+C+in+the+phosphorylation+of+rhodopsin.">Involvement of protein kinase C in the phosphorylation of rhodopsin.</a> The Journal of Biological Chemistry. 266 (27), 17725-17728 (1991).</li><li>Pinzon-Guzman, C., Zhang, S. S., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+protein+kinase+C+isoforms+are+required+for+rod+photoreceptor+differentiation.">Specific protein kinase C isoforms are required for rod photoreceptor differentiation.</a> The Journal of Neuroscience. 31 (50), 18606-18617 (2011).</li><li>Sokal, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+kinase+C+isozymes+responsible+for+the+phosphorylation+of+photoreceptor-specific+RGS9-1+at+Ser475.">Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475.</a> The Journal of Biological Chemistry. 278 (10), 8316-8325 (2003).</li><li>Rose, K., Walston, S. T., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+photoreceptor+cell+compartments+in+mouse+retina+for+protein+analysis.">Separation of photoreceptor cell compartments in mouse retina for protein analysis.</a> Molecular Neurodegeneration. 12 (1), 28 (2017).</li><li>Wright, A. F., Chakarova, C. F., Abd El-Aziz, M. M., Bhattacharya, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+degeneration:+genetic+and+mechanistic+dissection+of+a+complex+trait.">Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait.</a> Nature Reviews. Genetics. 11 (4), 273-284 (2010).</li></ol>

The authors declare that they have no competing interests.

Many retinal diseases affect rod photoreceptor cells, leading to rod death and, ultimately, complete vision loss37. A significant portion of the genetic and mechanistic origins of human retinal degeneration have been successfully recapitulated in numerous mouse models over the years. In that context, the ability to easily and selectively separate individual rod subcellular compartments from the small mouse retina would greatly enhance our understanding of the localized biochemical and molecular un...

Rod photoreceptor cells, tightly packed in the outermost layer of the neural retina, are an integral part of dim light vision. To function as faithful photon counters, rods utilize a G-protein-based signaling pathway, termed phototransduction, to generate rapid, amplified, and reproducible responses to single photon capture. This response to light ultimately triggers a change in the current at the plasma membrane and is subsequently signaled to the rest of the visual system1. As their name implies, each rod cell has a distinct rod-like shape and exhibits a highly polarized cellular morphology, consisting of an outer segment (OS), inner segment (IS), cell body (CB), and synaptic terminal (ST). Each subcellular compartment has specific protein machinery (membrane-bound and soluble), biomolecular features, and protein complexes that play crucial roles such as visual phototransduction, general housekeeping and protein synthesis, and synaptic transmission2,3.
Over 30 years ago, the light-dependent reciprocal movement of subcellular proteins, specifically transducin (away from the OS) and arrestin (towards the OS), was first observed4,5,6,7. Early on, this observed phenomenon was received with skepticism, due in part to immunohistochemistry&#39;s vulnerability to epitope masking8. In the early 2000s, stimulus-dependent protein translocation was confirmed by using a rigorous and arduous physical sectioning technique9. Serial tangential sectioning of the frozen flat-mounted rodent retinae followed by immunoblotting revealed that transducin9,10, arrestin11,12, and recoverin13 all undergo subcellular redistribution in response to light. It is believed that light-driven translocation of these key signaling proteins not only regulates the sensitivity of the phototransduction cascade9,14,15, but may also be neuroprotective against light damage16,17,18. Because light-driven protein transport in rods appears to be very significant to rod cell biology and physiology, techniques that permit the isolation of different subcellular compartments to determine protein distribution are valuable research tools.
Currently, there are a few methods aimed at isolating the rod subcellular compartments. However, these methods can be lengthy and difficult to reproduce, or require a sizable amount of retinal isolate. Rod outer segment (ROS) preparations via density gradient centrifugation19, for example, is commonly used to separate the ROS from retinal homogenate. This method is widely used for western blot, but the procedure is very time consuming and requires a minimum of 8-12 murine retinae20. On the other hand, serial tangential sectioning of frozen murine and rat retinae has been successfully implemented in isolating the OS, IS, CB, and ST9,11,13. However, this method is technically challenging due to the necessity of fully flattening the small and highly curved murine retina to align the retinal layers prior to tangential sectioning. Since there are a plethora of mouse models and transgenic mice recapitulating diseases of the visual system, the creation of a technique that reliably, quickly, and easily separates individual rod compartments holds promise in revealing the physiologic processes that occur in each specialized compartment and the mechanisms that underlie visual processes in health and disease.
To facilitate these investigations, we describe two peeling methods that isolate rod subcellular compartments more easily than current protocols. The first peeling method, adapted from a technique to expose fluorescently labeled bipolar cells for patch clamp recording21, employs cellulose filter paper to sequentially remove the ROS from a live, isolated murine retina (Figure 1). The second method, adapted from a procedure that isolates the three primary retinal cell layers from a chick22 and frog23 retina, utilizes adhesive tape to remove the ROS and rod inner segment (RIS) from a lyophilized retina (Figure 2). Both procedures can be completed in 1 h and are considerably user-friendly. We provide validation of the effectiveness of these two separation protocols for western blot by utilizing dark-adapted and light-exposed retinae from C57BL/6J mice to demonstrate light-induced translocation of rod transducin (GNAT1) and arrestin (ARR1). Moreover, using the tape peeling method, we provide additional evidence that our technique can be used to examine and address inconsistencies between protein localization data acquired by immunocytochemistry (ICC) and western blots. Specifically, our technique showed that: 1) the protein kinase C-alpha (PKC&#945;) isoform is present not only in bipolar cells, but also in murine ROS and RIS, albeit in low concentrations24,25, and 2) rhodopsin kinase (GRK1) is present predominantly in the isolated OS sample. These data demonstrate the effectiveness of our two peeling techniques for separating and quantifying specific rod and retinal proteins.

<table><tbody><tr><td>100 mL laboratory or media bottle equipped with a tubing cap adapter</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>100% O&lt;sub&gt;2&lt;/sub&gt; tank</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>1000mL Bottle Top Filter, PES Filter Material, 0.22 &amp;mu;m</td><td>Genesee Scientific</td><td>25-235</td><td></td></tr><tr><td>4X SDS Sample Buffer</td><td>Millipore Sigma</td><td>70607-3</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; and 5% CO&lt;sub&gt;2&lt;/sub&gt; tank</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Ames&amp;rsquo; Medium with L-glutamine, without bicarbonate</td><td>Sigma-Aldrich</td><td>A1420</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (99%, dihydrate)</td><td>Sigma</td><td>C-3881</td><td></td></tr><tr><td>Drierite (Anhydrous calcium sulfate, &gt;98% CaSO&lt;sub&gt;4&lt;/sub&gt;, &gt;2% CoCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>WA Hammond Drierite Co LTD</td><td>21005</td><td></td></tr><tr><td>Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm)</td><td>Falcon-Corning</td><td>08-757-100A</td><td></td></tr><tr><td>Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm)</td><td>Falcon-Corning</td><td>08-772F</td><td></td></tr><tr><td>Feather Scalpel (No. 10, 40 mm)</td><td>VWR</td><td>100499-578</td><td></td></tr><tr><td>Feather Scalpel (No. 11, 40 mm)</td><td>VWT</td><td>100499-580</td><td></td></tr><tr><td>KCl (99%)</td><td>Sigma</td><td>P-4504</td><td></td></tr><tr><td>Kimble Kontes pellet pestle</td><td>Sigma</td><td>z359971</td><td></td></tr><tr><td>Labconco Fast-Freeze Flasks</td><td>Labconco</td><td>N/A</td><td></td></tr><tr><td>LN&lt;sub&gt;2&lt;/sub&gt; (liquid nitrogen) + Dewar flask or similar vacuum flask</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M-9272</td><td></td></tr><tr><td>Milli-Q/de-ionized water</td><td>EMD Millipore</td><td>N/A</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (powder)</td><td>J.T. Baker</td><td>4062-01</td><td></td></tr><tr><td>NaCl (crystal)</td><td>EMD Millipore</td><td>Sx0420-3</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Amresco</td><td>0865</td><td></td></tr><tr><td>OmniPur EDTA</td><td>EMD</td><td>4005</td><td></td></tr><tr><td>OmniPur HEPES, Free Acid</td><td>EMD</td><td>5320</td><td></td></tr><tr><td>Parafilm M</td><td>Sigma-Aldrich</td><td>P7793</td><td></td></tr><tr><td>Reynolds Wrap Aluminum Foil</td><td>Reynolds Brands</td><td>N/A</td><td></td></tr><tr><td>Scotch Magic Tape (12.7 mm x 32.9 m)</td><td>Scotch-3M</td><td>N/A</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D67501</td><td></td></tr><tr><td>Spectrafuge mini centrifuge</td><td>Labnet International, Inc</td><td>C1301</td><td></td></tr><tr><td>Tissue incubation chamber (purchased or custom made)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Tris-HCl</td><td>J.T.Baker</td><td>4103-02</td><td></td></tr><tr><td>Triton X-100</td><td>Signma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>VirTis Benchtop 2K Lyophilizer or equivalent machine</td><td>SP Scientific</td><td>N/A</td><td></td></tr><tr><td>VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 &amp;mu;m)</td><td>VWR</td><td>28310-015</td><td></td></tr><tr><td>Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 &amp;mu;m)</td><td>Whatman/GE Healthcare</td><td>1001-090</td><td></td></tr><tr><td>Wide bore transfer pipet, Global Scientific</td><td>VWR</td><td>76285-362</td><td></td></tr></tbody></table>

two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

The present strategies were developed to provide relatively rapid and simple methods to isolate and analyze proteins among specific rod subcellular compartments for western blot analysis. We applied two sequential peeling techniques (Figure 1 and Figure 2) followed by immunoblotting to demonstrate that these methods could reliably be used to detect the known distribution of rod transducin (GNAT1) and arrestin (ARR1) in both dark- and light-adapted animals. To va...

Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

two-peeling-methods-for-isolation-photoreceptor-cell-compartments

Research

JoVE Journal

Neuroscience

3.1K Views.  University of Southern California. This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Video: Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Biochemistry

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Biology

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...