This work was supported by Agence Nationale de la Recherche (Jeunes Chercheuses/Jeunes Chercheurs to EFN), Fondation Voir et Entendre and Fondation Bettencourt Schueller (Young Investigator Grants to EFN), Centre National de la Recherche Scientifique (CNRS, permanent position for EFN), and The National Eye Institute of the National Institutes of Health (R01-EY13295 to SCF). Additionally, the Institut de la Vision is funded by Institut National de la Sant&#233; et de la Recherche M&#233;dicale, Universit&#233; Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and D&#233;partment de Paris.

This POS isolation experiment is time consuming and may require up to 12 hr to complete if POS are labeled before storage. The protocol has been adapted from a paper published by R.S. Molday and colleagues in 198731 and modified by S.C. Finnemann and colleagues in 199710.
Animals were handled according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Protocols were reviewed and approved ...

Three steps or conditions are crucial for an optimized purification: quality of gradient casting and delicate gradient tubes manipulation, keeping tissues chilled and in the dark until the collection step, strength of shaking of retinal homogenates to obtain proper POS isolation from the rest of the PR cell. If some issues arise in seeing the orange band properly, they are most likely due to one of the three reasons above (see also the second paragraph of the Results section). Some modification of certai...

In the retina, vision is triggered by isomerization of photosensitive molecules called opsins, before being transformed into a signal that can be transmitted between neurons up to the visual areas in the brain. These molecules are embedded in stacks of membranous disks resembling pancakes that constitute the outer segment portions of photoreceptor cells (PRs). Being subjected to constant exposure to light and therefore considerable levels of oxidative stress, PRs continuously renew their outer segments to limit potential oxidative damage. Photoreceptor outer segments are in close contact with apical microvilli of the neighboring retinal pigment epithelial (RPE) cells. RPE cells constitute the outer part of the blood-retinal barrier and ensure numerous tasks that are crucial for photoreceptors health and function1, such as quenching light rays via melanin pigments, re-isomerization of the photoreactive opsin component retinal, providing nutrients and growth factors, participating in PR metabolite disposal.
In addition, RPE cells eliminate spent POS and recycle their components, a daily occupation that is regulated by the circadian rhythm in mammalian retina2,3. The clearance of shed POS is absolutely necessary for PR survival. When it is completely abrogated, POS debris accumulate and PRs degenerate causing rapid vision loss4,5. If the rhythmic profile is lost and replaced by a constant activity, PR and RPE defects accumulate with age6. Therefore, it is very important to characterize the molecular regulation of RPE phagocytosis in vitro in order to understand phenotypes linked to its dysfunction. Interestingly, the molecular machinery in RPE cells is very similar to the one used by macrophages to clear apoptotic cells and both are dependent on recognition of exposed phosphatidylserine on phagocytic debris7-9. Still, RPE cells and macrophages regulate phagocytosis differently, as macrophages opt for immediate elimination of apoptotic cells at encounter time while RPE cells rhythmically engulf POS only once a day despite their permanent contact with outer segments. This suggests specific regulation mechanisms that are not yet fully understood.
Many of the molecules implicated in the RPE phagocytic machinery have been identified or validated thanks to the use of isolated POS and cell culture phagocytosis assays. The alphavbeta5 integrin receptor located at the RPE apical cell surface, in coordination with its ligand MFG-E8, binds specifically to POS10-12, which are then internalized via the MerTK tyrosine kinase receptor13-15. The CD36 scavenger receptor has been shown to participate in POS intake and influence its speed16,17, and might serve as a sensor of oxidized phospholipids at the POS surface18. Internalization needs the recruitment of F-actin cytoskeleton-associated proteins such as annexin 219, myosin II20 and myosin VIIA21,22. Native or oxidized POS in vitro are also utilized to understand aging phenotypes of RPE cells in vivo linked to accumulation of poorly digested oxidized POS23-28. The generation of RPE cells derived from stem cells has initiated a new application for isolated POS that are used to prove functionality of cells before they are transplanted to animals or patients29,30,27.
First described by Molday and colleagues in 198731, the protocol for isolation of bovine POS combines an ultracentrifugation step of retinal homogenates on continuous sucrose gradients with observation of the characteristic orange appearance of unbleached retinal photopigment (carrying 11-cis retinal). In the past 10 years, due to precautions taken in order to minimize risk of mad cow disease, use of porcine eyes has become increasingly prominent. The protocol described here shows how to obtain large amounts of POS from porcine or bovine eyes that can be aliquoted and stored for extended periods of time. This eliminates the need to prepare POS from rodent eyes, which requires using a large number of animals per POS preparation and assay32,33,21,22. In addition, details about POS labeling before storage using fluorescent molecules are given, to quantify and visualize POS in a simplified and comparable manner for some applications compared to labeling POS after the phagocytosis assay32,10. Therefore, these large stocks allow for reproducibility and ease of use in many different types of experiments.

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			<td height="21" style="height:21px;width:311px;">2-chamber gradient maker</td>
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			<td>gradient maker with 30-ml chambers</td>
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			<td height="21" style="height:21px;width:311px;">3-mm diameter silicone tubing</td>
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			<td>tubing for gradient casting</td>
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			<td height="21" style="height:21px;">PP Oak Ridge tubes</td>
			<td>Nalgene</td>
			<td>3119-0050</td>
			<td>30-mL centrifugation tubes&#160;</td>
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			<td height="21" style="height:21px;">Optima LE-80K&#160;</td>
			<td>Beckman Coulter&#160;</td>
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			<td>ultracentrifuge</td>
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			<td height="21" style="height:21px;">SW 32Ti swing rotor</td>
			<td>Beckman Coulter&#160;</td>
			<td>369694</td>
			<td>swing rotor for ultracentrifuge</td>
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			<td height="21" style="height:21px;">Avanti J-26 XP</td>
			<td>Beckman Coulter&#160;</td>
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			<td>centrifuge</td>
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			<td height="21" style="height:21px;">JA-25.50 rotor</td>
			<td>Beckman Coulter&#160;</td>
			<td>363058</td>
			<td>rotor for Avanti J-26 XP centrifuge</td>
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			<td>Life Technologies&#160;</td>
			<td>F-1906</td>
			<td>fluorescent dye</td>
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			<td style="width:291px;"></td>
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large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

The combination of the linear sucrose gradient and the ultracentrifugation allows the separation of the different components of the retinal suspension by density. Heavy larger retinal debris and RPE cells sink to or near the bottom of the gradient (Figure 2A). Lighter POS and lighter individual cells or cell debris from the retina migrate as separate bands to reach the top half of the gradient by the end of the centrifugation period. By keeping the eyes and the samples in the dark until after the centrif...

Watch this Scientific Journal Video about Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells at JoVE.com

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

large-scale-purification-porcine-or-bovine-photoreceptor-outer

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Immunology and Infection

11.8K 조회수.  INSERM, U968. This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C.