Name: large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells
Uploaded: 2014-12-12T13:00:00
Description: 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

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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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

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

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High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

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Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

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Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

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Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...