This work was supported by the National Eye Institute (NEI), and National Eye Institute (NEI) fund R01 EY029751-04. We would like to thank Dr. Pamela Martin for her help in our initial stages of RPE isolation.

Animals were used as per the guidelines of Oakland university IACUC animal protocol number 21063 and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Solution preparation
<ol>
	<li>Prepare the complete RPE cell culture media by supplementing Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with 25% Fetal Bovine Serum (FBS), 1.5% penicillin/streptomycin, and 0.02% gentamicin.</li>
	<li>Prepare Enzyme A by supplementing 741 &#181;L of Hank&#39;s Balanced Salt Solution (HBSS) with 127 &#181;L of Collagenase stock solution and 132 &#181;L of Hyaluronidase stock solution. This makes a final volume of 1 mL, which can treat four eyes.
	<ol>
		<li>Prepare the Collagenase stock solution by dissolving 1 mg of Collagenase from Clostridium histolyticum in 41 mL of HBSS (19.5 units /mL).</li>
		<li>Prepare the Hyaluronidase stock solution by dissolving 1 mg of Hyaluronidase from bovine testes in 18.4 mL of HBSS (38 units /mL).</li>
	</ol>
	</li>
	<li>Prepare Enzyme B by adding two parts of 0.25% Trypsin-EDTA to three parts of HBSS. Note that the total volume is dependent on the number of eyes dissected; 1 mL of Enzyme B can be used to treat four eyes.</li>
</ol>
2. Enucleation
<ol>
	<li>Ethically euthanize the mouse using carbon dioxide (CO2) inhalation according to IACUC standards21.
	<ol>
		<li>Briefly, place the animal(s) in the CO2 chamber to introduce 100% CO2 with a fill rate of 30%-70% of the chamber volume per minute with CO2 to achieve unconsciousness within 2 to 3 min, with minimal distress to the mice.</li>
		<li>Confirm death (lack of respiration and faded eye color), then move the mouse from the cage to a clean sterilized surgical area (scrubbed with alcohol) equipped with sterilized surgical equipment.</li>
	</ol>
	</li>
	<li>Place the mouse on a sterile underpad.</li>
	<li>Enucleate the eyes by pressing 5/45 style tweezers on the orbital area to induce proptosis, followed by moving the tweezers to the posterior of the eye. Extract the eyes, with the optic nerve intact, by pulling the eyes gently from the orbital cavity.</li>
	<li>Using forceps, submerge the eyes in a 2 mL microcentrifuge tube containing 1mL of 5% Povidone-Iodine. Incubate the eyes at room temperature for 2-3 min.</li>
	<li>Remove the eyes from the povidone-iodine solution and place them in a Petri dish. Using a sterile transfer pipette, wash the eyes with HBSS until the orange color of the povidone-iodine is gone. This takes around two or three washes.</li>
	<li>Place the eyes in a new 60 mm Petri dish and submerge them in cold (2-8 &#176;C) complete RPE cell culture media prepared in step 1.1.</li>
</ol>
3. Dissection
<ol>
	<li>Under a surgical microscope, use M5S style tweezers and Vannas scissors to remove the connective tissue and extraocular muscles. 
	NOTE: The dissection is done under a tissue hood in a completely sterile environment. However, for video purposes, the dissecting microscope was placed outside the hood to achieve a higher quality demonstration/filming. Placing a small piece of lint-free tissue paper under the eye improves stability during dissection. The eyes can then be held temporarily (not for more than 1 hour) in the cold RPE cell culture media. However, it is preferable to proceed directly to the next step immediately after cleaning the eyes.</li>
	<li>Transfer the eyes to a 2 mL microcentrifuge tube containing 1 mL of Enzyme A solution for every four eyes. Then, incubate the eyes in an incubator at 37 &#176;C for 40 min.</li>
	<li>Remove the eyes from the incubator and microcentrifuge tube. Then, transfer them to a new microcentrifuge tube containing 1 mL of Enzyme B solution for every four eyes. Incubate the tube in an incubator at 37 &#176;C for 50 min.</li>
	<li>Place the eyes in a new 60 mm suspension culture dish containing 10 mL of complete RPE cell growth media. Then incubate the eyes at 4 &#176;C for 30 min.</li>
	<li>Place the dish under a surgical microscope and begin dissecting.</li>
	<li>Remove the anterior part of the eye (cornea, iris, and lens) from the posterior part of the eyecup (posterior retina).
	<ol>
		<li>Grasp the eye with M5S style forceps and make an incision in the ora serrata section (most anterior part of the retina where the light blue ends) with a #11 blade or the needle of a syringe.</li>
		<li>Grasp the inner part of the incision with one pair of forceps, and grasp the cornea with another pair of forceps. Begin to slowly pull or tear the cornea from the retina. Be sure to tear in small increments and move down the tear while removing the cornea, to ensure that the RPE remains mostly intact and results in a cleaner tear. 
		NOTE: Once the cornea is completely detached, the iris and lens can be seen.</li>
		<li>Separate both the cornea and iris.</li>
	</ol>
	</li>
	<li>Separate the lens from the eyecup with gentle compression of the posterior half of the eye.</li>
	<li>To remove the neural retina from the RPE layer, grasp the outer layer of the eyecup with one pair of tweezers and position the arm of a second pair of tweezers between both the RPE and the neural layers. From there, gently pull or scoop the neural retina away from the RPE layer. Discard the neural retina. 
	NOTE: In the video, the neural retina and lens are removed together. However, for beginners, it will be easier to remove each separately. What remains is the RPE, choroid, and sclera.</li>
	<li>Discard the cornea, iris, lens, and neural retina. Move the remaining eyecup to a new area on the dish free of debris.</li>
	<li>To separate the RPE from the choroid and sclera, gently scrape the inside of the eyecup with 5/45 style tweezers to ensure the separation of the RPE cells. The RPE cells appear cloudy when suspended in the complete RPE cell growth media.</li>
	<li>Aspirate the cells using a 3.2 mL sterile transfer pipette and transfer to a new 15 mL centrifuge tube containing complete RPE cell growth media.</li>
</ol>
4. Centrifugation
<ol>
	<li>Place the 15 mL centrifuge tube containing primary RPE cells inside a centrifuge and centrifuge the cells at 300 x g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the pellet with 10 mL of complete RPE growth media. The complete RPE growth media is more specific for RPE growth than other contaminating cells, such as M&#252;ller cells or choroidal endothelial cells. M&#252;ller cells require high glucose media, while choroidal endothelial cells require specific growth factors. By changing the media and passaging the culture, only the RPE cells will continue to grow.</li>
</ol>
5. Cell culture
<ol>
	<li>Plate the resuspended primary RPE cells onto a sterile 100 mm Petri dish and transfer them to an incubator (after verifying the purity by staining cells with specific RPE/M&#252;ller cell markers, as indicated in Figure 1E-N). Incubate the cells at 37 &#176;C and 5% CO2. 
	NOTE: The markers used are mentioned in the Table of Materials. This step is performed after the culture is established.</li>
	<li>Incubate the cells for about 5 days to grow. During this time, do not change the media, disturb the cells, or move the cell culture plate (this is a critical step in the protocol; after isolation, the cells should be incubated and not be disturbed for 5 days). 
	NOTE: The number of cells depends on the number of eyes used for the isolation. A greater number of eyes isolated will yield a greater number of cells. RPE isolation from two eyes will yield ~ 440,000-880,000 cells, or 5%-10% of the 100 mm Petri dish, which can hold 8.8 million cells.</li>
</ol>
6. Passaging
<ol>
	<li>Aspirate out the media and wash with 2 mL of PBS. Then aspirate out the PBS.</li>
	<li>Add 4 mL of 0.25% Trypsin-EDTA (1x), or enough to cover the base of the dish. Incubate for 5-10 min at room temperature.</li>
	<li>Add 8 mL of pre-warmed RPE media or twice the volume of how much trypsin was used in step 6.2. Then collect the cells and place them in a 15 mL centrifuge tube.</li>
	<li>Centrifuge at 300 x g for 7 min at room temperature. Aspirate the supernatant and resuspend the pellet in 10 mL of complete RPE cell culture media. Plate the whole suspension in a sterile 100 mm Petri dish. 
	NOTE: The cells can be passaged up to four or five times18. However, the most consistent experiment results are found after two or three passages. After two to four passages, the cells changed their shape (as shown in Figure 1D) to be more elongated, accumulated in the middle of the plate, stopped growing, and detached.</li>
	<li>Use immunofluorescence protocols, as per the previously published methods8,14,15,16, to stain and validate the RPE specificity.</li>
</ol>
7. Immunofluorescence
NOTE: Immunofluorescence was performed as per the previously published methods8,14,15,16,17,18 to stain and validate the RPE specificity. The staining of primary RPE cells was typically done at passages P1 and P2. Here, a brief overview of the immunofluorescent protocol is presented.
<ol>
	<li>Seed the cells on a cell culture chamber slide (30,000 cells per well for the 8-well chamber slide).</li>
	<li>Culture the cells in complete RPE cell culture media at 37 &#176;C and 5% CO2 for 24-48 h.</li>
	<li>When the cells are 80%-90% confluent, aspirate out the media and wash the chamber slide with 1x PBS.</li>
	<li>Fix the slide with 4% paraformaldehyde for 10-15 min.</li>
	<li>Aspirate out the 4% paraformaldehyde and then block with blocking solution (see Table of Materials).</li>
	<li>Aspirate out the blocking solution and add the primary antibody, RPE65, and incubate for three hours at 37 &#176;C (with an antibody concentration ranging from 1:200-1:250 in blocking buffer, at a volume of 150-200 &#181;L/slide). Then, wash three times with PBS/TX-100 (5-10 min each wash).</li>
	<li>Aspirate out the primary antibody and add the secondary antibody (with an antibody concentration 1:1,000 in blocking buffer, at a volume of 150-200 &#181;L/slide). Incubate for one hour at 37 &#176;C.</li>
	<li>Aspirate out the secondary antibody. Wash three times with PBS/Trtion X-100 (5-10 min each wash).</li>
	<li>Add a drop of DAPI mounting medium to label the nuclei and place a coverslip over the slide. Ensure that there are no bubbles beneath the coverslip.</li>
	<li>Then, take images with a fluorescent microscope, accompanied by a high-resolution microscope (HRM) camera and image processing software (see Table of Materials).</li>
</ol>

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The authors have no conflicts of interest to declare that are relevant to the content of this article.

The current protocol is a reported, modified, and simplified detailed procedure for RPE isolation from mouse eyes. The protocol includes enucleation, dissection, collection, seeding, culture, and characterization of RPE cells isolated from mouse eyes.
There are some limitations and critical steps that must be fulfilled for successful RPE isolation, such as mice age, the number of eyes dissected, the size of the tissue culture plate or dish, and cautions after seeding, storage, and passaging. To be able to passage the isolated cells up to three or four times, the best mice ages are between 18-21 days. To have a reasonable number of isolated cells that are able to grow and multiply, at least two eyes are needed for single isolation. The number of cells resulting in isolation is proportional to the number of eyes dissected (~220,000 cells/eye). A sterile 100 mm Petri dish/flask is recommended to obtain a larger number of cells in an early passage (P0). After seeding, cells should not be disturbed by moving the tissue culture plate from the incubator or by changing the media for at least 5 days.&#160;Also, one of the limitations ofthis technique is the storage and passaging of the isolated cells. Cells cannot be stored (freezed and rethawed) and can only be passaged three or four times. After passage 4, cells start to change their size and shape to become elongated (spindle shape) with a marked decrease in cell pigmentation (Figure 1D).
This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20,24 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of M&#252;ller cells was achieved by staining isolated cells with a specific cell marker for M&#252;ller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn&#39;t require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.
Primary RPE isolation is a technique that could be an important tool to study underlying mechanisms and pathogenesis, and for proposing new therapeutic applications for different eye diseases. The current technique enabled studying the changes in RPE cell structure and function and their impact on the outer BRB in age-related macular degeneration8. Moreover, it helped in studying the changes in RPE cells isolated from wild-type C57BL6 mice and different genetically modified mice, as well as testing the different pharmacological compounds seeking a therapeutic target for AMD14,16.
In conclusion, many available published protocols demonstrated primary mouse RPE isolation. The presented protocol is a simplified procedure resulting from modifying some existing protocols, using material, methods, and equipment that are available in most research labs to isolate primary RPE cells from mouse eyes.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63543">Isolation of Primary Mouse Retinal Pigmented Epithelium Cells</a>. The Discussion and References sections were updated.
The third paragraph of the Discussion&#160;section was updated from:
This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of M&#252;ller cells was achieved by staining isolated cells with a specific cell marker for M&#252;ller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn&#39;t require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.
to:
This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20,24 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of M&#252;ller cells was achieved by staining isolated cells with a specific cell marker for M&#252;ller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn&#39;t require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.
In the References section, a 24th reference was added:
<ol start="24">
	<li>Promsote, W., Makala, L., Li, B., Smith, SB., Singh, N., Ganapathy, V., Pace, B.S., Martin, P. Monomethylfumarate induces &#947;-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina. Invest Ophthalmol Vis Sci. 55(8):5382-93 (2014).</li>
</ol>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/63543">Isolation of Primary Mouse Retinal Pigmented Epithelium Cells</a>. The Discussion and References sections were updated.

Erratum: Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Retinal pigmented epithelium (RPE) cells are located between the choroid and the neural retina, forming a simple monolayer of cuboidal cells that lies behind the photoreceptor (PR) cells1. The RPE plays a critical role in maintaining a healthy environment for PR cells, mainly by reducing the excessive accumulation of reactive oxygen species (ROS) and consequent oxidative damage1.&#160;RPE cells oversee many functions, such as the conversion and storage of retinoids, the absorption of scattered light, fluid and ion transport, and phagocytosis of the shed PR outer segment membrane2,3. Alterations in the RPE (morphology/function) can impair their function leading to retinopathy and this is a common feature shared by many ocular disorders4. Many ocular diseases are associated with alterations in the morphology and function of RPE cells, including some genetic diseases such as retinitis pigmentosa, Leber congenital amaurosis, and albinism4,5,6, as well as age-related ocular disorders such as diabetic retinopathy (DR) and age-related macular degeneration (AMD)7,8. Human cells are the most desirable, thus it would be ideal to study RPE disorders in primary human RPE cells for forming RPE monolayers. However, ethical matters and the limited availability of human donors due to the fact that most of these disorders lead to morbidity9, but not necessarily mortality10, thereby prevent the isolation of primary human RPE cells. This makes culturing RPE cells from nonhuman animal donors a preferred alternative. Rodents, particularly mice, are considered a great model for studying different ocular diseases since transgenic technology is more extensively established in these species11. Even though the use of cultured primary RPE cells offers many advantages, it has been difficult to maintain growing cells for many passages, or to store and reuse the cells. The main limitation of this protocol is the mice&#39;s age; mice that are used for RPE isolation should be of a very young age (18-21 days old is optimal) as it has been difficult to culture RPE cells from adult mice11,12,13. RPE cells can be isolated from mouse eyes at any age, however up to four cell passages was only successful with young mice (18-21 days old). RPE isolation from mouse retinas, using both C57BL6 mice and transgenic mice with deletion of the N-methyl-D-aspartate receptors (NMDARs) at the RPE cells, was performed to study the effect of elevated amino acid homocysteine on the development and progression of AMD14. In addition, isolated primary RPE cells helped in proposing a therapeutic target for AMD by inhibition of the NMDARs at RPE cells14. There are some NMDAR blockers that are approved by the Food and Drug Administration (FDA) and are currently used to treat moderate to severe confusion (dementia) related to Alzheimer&#39;s disease (AD), such as memantine16, which could be a potential therapeutic target for AMD14. Furthermore, isolated primary mouse RPE cells were used for the detection of inflammatory markers and the proposed induction of inflammation as an underlying mechanism for homocysteine-induced features of AMD and AD using a genetically modified mouse (CBS), which presents a high level of homocysteine16,17.
This protocol was used to isolate RPE cells from both wild-type C57BL/6 mice and transgenic mice developed in our lab as a simplified adaptation of other published isolation protocols13,18,19 to reach an easily applicable and reliable protocol. There is no sex preference in this protocol. While mice ages are critical for the isolation process, young, aged mice (18-21 days old) and older mice at any age (up to 12 months) were used for RPE isolation. However, we noticed that the RPE cells isolated from the young-aged mice lived longer, and up to four passages could be performed. The RPE cells isolated from older mice could be passaged once or twice, then they would stop growing at a normal rate and change their shape to be more elongated (fibroblast-like cells). Loss of pigmentation and decreased adhesion to the tissue culture plate followed by detachment was also observed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beaker : 100mL</td>
			<td>KIMAX</td>
			<td>14000</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C7657-25MG</td>
			<td>For working enzyme, A</td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes :3.2mL Sterile</td>
			<td></td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>&#160;gibco&#160;</td>
			<td>11330</td>
			<td>Media to grow RPE cells&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>gibco</td>
			<td>26140079</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent Solution</td>
			<td>gibco</td>
			<td>15750-060</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo Scientific</td>
			<td>88284</td>
			<td>For working enzymes (A&#38;B)&#160;</td>
		</tr>
		<tr>
			<td>Heracell VISO 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes</td>
			<td>Sigma-Aldrich</td>
			<td>H3506-500MG</td>
			<td>For working enzyme A</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL</td>
			<td>B-D</td>
			<td>309577</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tube: 2 mL</td>
			<td>Grainger</td>
			<td>11L819</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-RPE65 antibody&#160;</td>
			<td>Abcam, Cambridge, MA, USA</td>
			<td>ab78036</td>
			<td>For IF staining&#160;</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>gibco</td>
			<td>15140-122</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Positive Action Tweezers, Style 5/45</td>
			<td>Dumont</td>
			<td>72703-DZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Iris Standard Straight 11.5cm</td>
			<td>GARANA INDUSTRIES</td>
			<td>2595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall St8 Centrifuge</td>
			<td>ThermoScientific</td>
			<td>75007200</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 305 Microscope</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade, #11, Stainless Steel</td>
			<td>Bard-Parker</td>
			<td>371211</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension Culture Dish 60mm x 15mm Style</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish : 100x20mm style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 15mL</td>
			<td>Midsci</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 50mL</td>
			<td>Midsci</td>
			<td>C50R</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (1x) 0.25%</td>
			<td>gibco</td>
			<td>2186962</td>
			<td>For working enzyme B</td>
		</tr>
		<tr>
			<td>Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips</td>
			<td>Dumont</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL</td>
			<td>Electron Microscopy Sciences Dumont</td>
			<td>50-241-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Underpads, Moderate : 23&#34; X 36&#34;</td>
			<td>McKesson</td>
			<td>4033</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioImager Z2</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen Blue 2.6</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Validation of the specificity, purity, and barrier function/formation of isolated RPE cells 
The isolated cells were examined under a light microscope to verify their viability, morphology, and pigmentation. Images from P0 and P1 (Figure&#160;1A,B) and images from P0 and P4 were captured (Figure 1C,D) to show the changes in the cells; shape, size, and pigmentation as the passages proceeded to the fourth passage (black arrows are pointing to the isolated RPE cells in P4).&#160;An antibody for the RPE65 protein was used&#160;to verify the identity of the isolated cells.&#160;Immunofluorescence staining of the&#160;isolated cells was performed with an&#160;antibody against RPE65, followed by&#160;examination under the fluorescent&#160;microscope (Figure 1E,F: low&#160;magnification, and Figure 1G,H: high&#160;magnification, showing positively stained&#160;RPE cells in green and a nuclear marker,&#160;DAPI, in blue). RPE65 is an&#160;isomerohydrolase expressed in the RPE.&#160;It is essential for the regeneration of the visual pigment required for rods- and cone-mediated vision21 .To validate the barrier function of the isolated 
cells, both cytoskeletal protein F-actin and tight junction protein ZO-1 immunofluorescence staining8 for the isolated cells were used (Figure 1I,J: green ZO-1 and blue nuclear staining). and Figure 1K,L: red F-actin and blue nuclear staining). However, the typical cobblestone shape of the RPE is very evident when cells are fully confluent.
Negative control was used in the experiments where the secondary antibody was added to the RPE cells without adding the primary antibody, to make sure the specificity of the antibody and the resultant color is specific to the antibodies that were used (not shown).
To validate the purity and contamination of the isolated RPE by M&#252;ller cells, immunofluorescence staining of the isolated cells with a specific cell marker for M&#252;ller cells, vimentin, was used (green and blue for nuclear staining as shown in Figure 1M,N) using M&#252;ller cells as a positive control. Isolated RPE cells showed negative staining for vimentin.
The isolated cells were examined under a light microscope to verify their viability, morphology, and pigmentation. Images from P0 and P1 (Figure 1K,L) and images from P0 and P4 were captured (Figure 1M,N) to show the changes in the cells&#39; shape, size, and pigmentation as the passages proceeded to the fourth passage (black arrows are pointing to the isolated RPE cells in P4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63543/63543fig01v2.jpg" /> 
Figure 1: RPE isolation validation. (A) Light microscope for RPE passage zero (P0). (B) Light microscope for RPE passage one (P1). (C) Morphology characterization at P0. (D) Morphology characterization at P4. (E) Immunostaining for RPE65 at low magnification. (F) Immunostaining for RPE65 with nuclear staining with DAPI. (G)&#160;Immunostaining for RPE65 at high magnification. (H) Immunostaining for RPE65 with nuclear staining with DAPI at high magnification. (I) ZO-1 staining for RPE cells. (G) ZO-1 staining for RPE cells with nuclear staining with DAPI. (K) F-actin staining for RPE cells. (L) F-actin staining for RPE cells with nuclear staining with DAPI. (M) Vimentin staining for RPE cells. (N)&#160;Vimentin staining for M&#252;ller cells as a positive control (RMc-1) with nuclear staining with DAPI. Scale bars are equal to 300 &#181;m, 20 &#181;m, 300 &#181;m, 300 &#181;m, 50 &#181;m, 10 &#181;m, 50 &#181;m, 50 &#181;m, and 50 &#181;m, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63543/63543fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Primary Mouse Retinal Pigmented Epithelium Cells at JoVE.com

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

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 ...

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4.8K Views.  Oakland University. 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.

Video: Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

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 ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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 and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...