This work was supported by National Eye Institute (NEI),The National Eye Institute (NEI) fund R01 EY029751-04. We would like to acknowledge Dr. Sylvia B. Smith as this protocol was modified version based on her protocol of M&#252;ller cell isolation.

All experiments with animals conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were done following our animal protocol approved by the Institute for Animal Care and Use Committee (IACUC) and Oakland University policies (protocol number 2022-1160)
1. Media and solution preparation
<ol>
	<li>Prepare M&#252;ller cell eye solution by supplementing the Dulbecco Modified Eagle Medium (DMEM, details in the material table) with penicillin/streptomycin at the dilution of 1:1000. For instance, for 10 mL of DMEM, add 10 &#181;L of penicillin/streptomycin. 
	NOTE: This is a simple serum-free media prepared. Also, warm the complete media in a 37 &#176;C water bath before using it for cell culture work.</li>
	<li>Prepare complete primary M&#252;ller cell growth media by supplementing DMEM with 10% Fetal Bovine Serum (FBS, heat-inactivated), and 1% penicillin/streptomycin. For example (500 mL of DMEM media + 50 mL of FBS + 5 mL of penicillin/streptomycin).</li>
	<li>Prepare a 0.05% Trypsin-EDTA solution with Collagenase IV at a concentration of 200 U/mL (Trypsin-EDTA is used to dissolve the calculated amount of Collagenase IV powder to reach the needed concentration).</li>
</ol>
2. Enucleation
<ol>
	<li>Ethically euthanize the mouse using CO2 inhalation following the IACUC guidelines.
	<ol>
		<li>Briefly, place the animal(s) in the CO2 chamber to introduce 100% CO2 at a fill rate of 30-70% of the chamber vol/min with CO2 to achieve the goal of rapid unconsciousness within 2-3 min with minimal distress to the mice.</li>
		<li>Since mice are at a young age (~10 days), perform cervical dislocation as a secondary method to ensure proper euthanasia.</li>
	</ol>
	</li>
	<li>After sterilizing the working area with 70% ethanol, place the mouse on a sterile under-pad.</li>
	<li>Extract the eyes by placing the tweezers&#39; arms at the back of the eye, gently pulling from the orbital area, and enucleating the eyes by applying pressure with 5/45 style tweezers to the orbital area to cause proptosis. 
	NOTE: Sterilize the dissection tools with 70% ethanol followed by phosphate buffer saline prior to the dissection.</li>
	<li>Ensure that the optic nerve is still connected to the eye by enucleating the eye slowly. Make sure to pull the optic nerve (visually identified as a white cord) behind the eye. 
	NOTE: This step does not need to be performed under a microscope, and can be performed on a sterilized bench.</li>
</ol>
3. Treatment of the enucleated eyes
<ol>
	<li>Cover a 15 mL centrifuge tube with aluminum foil and fill the tube with 10 mL of the M&#252;ller cell eye solution.</li>
	<li>Place the dissected eyes in the 15 mL tube containing M&#252;ller cell eye solution and allow them to sit overnight, or about 18 h, at room temperature.</li>
	<li>After 18 h, decant the eye solution and briefly rinse the eyes with 2-3 mL of warmed (37 &#176;C) phosphate buffered saline (PBS).</li>
	<li>Decant the PBS and move the eyes to a 100 mm Petri dish containing 10 mL of trypsin solution. (prepared in step 1.3). 
	NOTE: Trypsin is used on the whole enucleated eye, acting as a dissociative agent to facilitate cell separation from the retinal layers during dissection. The dissected retina has many cells, including RPE cells, which are sticky and most likely to contaminate M&#252;ller cell culture14. Microscopic examination clearly demonstrated that after 5 days of isolation (during the first media change), RPE cells detached from the plate and died13.The media used for RPE isolation is DMEM/F-12, with a different composition than DMEM (used in M&#252;ller cells isolation), containing sodium pyruvate and low glucose.</li>
	<li>Place the dish in an incubator and incubate for 1.5 to 2 h at 37 &#176;C and 5% CO2.</li>
	<li>After incubating, transfer the eyes to a 60 mm Petri dish containing roughly 5 mL of complete primary M&#252;ller cell growth media.</li>
</ol>
4. Dissection
NOTE: This procedure must be carried out within the sterile environment of the culture hood. Hence, it is essential to thoroughly sterilize the hood surfaces with 70% alcohol, along with all the tools and the dissection microscope. It is worth noting that the video was recorded outside the hood for better visibility and clearer demonstration purposes.
<ol>
	<li>Place a small piece of sterilized lab-grade tissue in the dish to help stabilize the eyes during dissection. Place the eyes under a dissecting microscope to start the dissection. 
	NOTE: The use of lab-grade tissue facilitates the maneuvering of eyes in a fluid-filled dish (complete media), ensuring precise dissection.</li>
	<li>Use Vannas scissors and M5S-style tweezers to remove the connective tissue, extraocular muscles, and optic nerve.
	<ol>
		<li>Grasp the eye with M5S style tweezers and make an incision in the ora serrata section with Vannas scissors or a needle of a syringe. 
		NOTE: There is a light blue circular line between the anterior and posterior part of the eye called ora serrata, which can be used as a landmark for accurate direction during dissection.</li>
		<li>Grasp the incision with M5S style tweezers and pull or tear the cornea from the posterior part of the eye. Pull in small increments to ensure the integrity of the retina remains intact. 
		NOTE: These steps are to remove the anterior part of the eye (Cornea, Iris, and Lens) from the posterior part of the eye cup (neural and pigmented retinas).</li>
		<li>To maintain the integrity of the retinal layers, pull gently and remove the retinal pigmented epithelium from the neural retina. The neural retina should be free of any black specks to ensure the purity of the isolation as much as possible, as the RPE layer is usually sticky and attached to the neuronal retina.</li>
		<li>Cut the dissected neural retina into small pieces before collecting it in a plate to ensure a homogenous spread of the cells on the plate.</li>
	</ol>
	</li>
	<li>Plate the neural retinas in a T25 flask containing 4 mL of complete primary M&#252;ller cell growth media.</li>
	<li>Finally, place the flask in an incubator and incubate at 37 &#176;C and 5% CO2.</li>
</ol>
5. Culturing of primary M&#252;ller glial cells
<ol>
	<li>Do not move the flask for 3-5 days to promote cell growth.</li>
	<li>Change the media after the fifth day and every other day thereafter until the cells are 90% confluent.</li>
</ol>
6. Passaging of primary M&#252;ller glial cells
<ol>
	<li>Aspirate the M&#252;ller cell culture media, followed by 2 mL of wash in PBS, and aspirate out the PBS. Add 2 mL of 0.25% Trypsin-EDTA (1x), or enough to cover the plate.</li>
	<li>Incubate for 5-10 min at room temperature.</li>
	<li>Ensure the cells are detached from the plate under the microscope. Then, collect the cell suspension and add 4 mL of pre-warmed (37 &#176;C) complete primary M&#252;ller cell growth media (prepared in step 1.2) to the collection tube.</li>
	<li>Centrifuge for 7 min at 300 x g. After aspirating the supernatant, resuspend the pellet in 10 mL of complete primary M&#252;ller cell growth media. Because the cell number can vary according to the number of eyes dissected, therefore, it is better to count the cells before seeding and incubation. The recommended seeding density is 3.0 x 106 cells in a T75 flask. 
	NOTE: Cells can be passaged up to 4-5 times. However, the most consistent experiment results are found after 1-2 passages.</li>
</ol>
7. Immunofluorescence
NOTE: Use the immunofluorescence protocol&#160;to stain and validate M&#252;ller cell specificity. Here is a brief overview of the immunofluorescent protocol. This step is performed after the first passage12.
<ol>
	<li>Seed the cells into a Cell Culture Chamber Slide (30,000 cells per well for an 8-well chamber slide).</li>
	<li>Culture the isolated cells for 24-48 h in a complete M&#252;ller cell culture medium (prepared in step 1.2) at 37 &#176;C and 5% CO2.</li>
	<li>Aspirate out the media and wash the chamber slide with 250-300 &#956;L of 1x PBS on each well when the cells are 80-90% confluent.</li>
	<li>Fix the slide for 10 to 15 min in 250-300 &#956;L of 4% paraformaldehyde on each well, followed by washing with PBS/TX-100 (5 min each/3x).</li>
	<li>Add blocking buffer for 1 h at 37 &#176;C (see the Table of Materials).</li>
	<li>Aspirate the blocking solution, then add the primary antibodies specific for M&#252;ller cells to confirm the purity of the isolated cells using glutamine synthetase and vimentin5,12. Incubate for 3 h at 37 &#176;C (using an antibody concentration of 1:100-1:200 in blocking buffer in a volume of 150-200 &#956;L/slide). Wash the slide with PBS/TX-100 washes (5 and 10 min each). 
	NOTE: Those antibodies were chosen as they are hallmarks for identifying M&#252;ller cells and ensuring the success and purity of the M&#252;ller cell culture.</li>
	<li>Add the secondary antibody (with an antibody concentration ranging from 1:1000 in blocking buffer in a 150-200 &#956;L/slide) and incubate for 1 h at 37 &#176;C. Then, wash with PBS/Triton X-100, three times (5-10 min each).</li>
	<li>Apply a drop of DAPI mounting medium to label the nuclei before covering the slide with a coverslip. Avoid air bubbles when placing the coverslip.</li>
	<li>Use a fluorescent microscope to check the slide and capture images (see Table of Materials). 
	NOTE: Cells are located with DAPI at ex/em 359/461 at 10x magnification to locate cells; higher magnification can capture cells. Other wavelengths would depend on the chosen color. Green color (GFP)- ex/em 488/510. Red color (Cy3)- ex/em 555/569.</li>
</ol>

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J., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cell-dependent+regeneration+of+the+neural+retina:+An+overview+across+vertebrate+model+systems.">M&#252;ller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.</a> Dev Dyn. 245 (7), 727-738 (2016).</li><li>Kobat, S. G., Turgut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+M&#252;ller+cells.">Importance of M&#252;ller cells.</a> Beyoglu Eye J. 5 (2), 59-63 (2020).</li><li>Liu, X., Tang, L., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+M&#252;ller+cell+isolation+and+culture.">Mouse M&#252;ller cell isolation and culture.</a> Bio Protoc. 7 (15), e2429 (2017).</li><li>Bringmann, A., 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=M&#252;ller+cells+in+the+healthy+and+diseased+retina.">M&#252;ller cells in the healthy and diseased retina.</a> Prog Ret Eye Res. 25 (4), 397-424 (2006).</li><li>de Melo Reis, R. A., Ventura, A. L. M., Schitine, C. S., de Mello, M. C. F., de Mello, F. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J of Neurobiol. 44 (3), 289-307 (2000).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler, J. S., Sinha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+from+the+mouse+retinal+pigment+epithelium.">Primary cell cultures from the mouse retinal pigment epithelium.</a> J of Vis Exp. (133), 56997 (2018).</li><li>Sarthy, P. V., Bunt, A. 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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=Progesterone+receptor+membrane+component+1+(PGRMC1)+expression+in+murine+retina.">Progesterone receptor membrane component 1 (PGRMC1) expression in murine retina.</a> Curr Eye Res. 41 (8), 1105-1112 (2016).</li><li>Moustafa, M., 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=12-HETE+activates+M&#252;ller+glial+cells:+The+potential+role+of+GPR31+and+miR-29.">12-HETE activates M&#252;ller glial cells: The potential role of GPR31 and miR-29.</a> Prostag Oth Lipid M. 171, 106805 (2024).</li><li>Pereiro, X., Ruzafa, N., Acera, A., Urcola, A., Vecino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+method+to+isolate+and+culture+adult+porcine,+rats+and+mice+m&#252;ller+glia+in+order+to+study+retinal+diseases.">Optimization of a method to isolate and culture adult porcine, rats and mice m&#252;ller glia in order to study retinal diseases.</a> Front Cell Neurosci. 14, 7 (2020).</li></ol>

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

The isolation of primary retinal pigmented epithelium (RPE) from mice was previously documented by our lab. This manuscript describes a detailed demonstration protocol for primary M&#252;ller cells isolation. This procedure involves enucleation, treatment, dissection, collection, seeding, culture, and characterization of M&#252;ller cells isolated from mouse eyes. It is based on a previously successful protocol found in earlier publications&#160;and our modified protocol that we used in a recent publication12,13. While the steps are straightforward, successful M&#252;ller cell isolation requires adherence to specific restrictions and essential requirements. After numerous trial and error attempts to optimize this protocol, multiple factors influencing its success were identified.
The ideal mouse age, as observed in our experiments, was found to be between 5-11 days in order to grow the isolated cells up to four passages. The culture is also influenced by how many eyes are isolated and cultured. For single isolation, at least two eyes are required to obtain sufficient number of cells to be able to grow and multiply. Another important aspect is the duration between enucleation and plating; therefore, this step needs to be performed relatively quickly. It was observed that prolonged manipulation of the eye tissue has a detrimental effect on the cells&#39; ability to adhere to the flask and multiply. To make sure that the proximity of isolated cells is close enough to encourage proliferation, we advise using a T25 flask. Once the first passage has been made, the cells can be plated on a T75. Also, cells should not be disturbed after isolation and seeding by removing the tissue culture flask from the incubator or by switching the media for at least 5 days. Finally, cells can only be passed about four times before they stop adhering. Although this procedure is straightforward and easy to perform, it has some limitations including the mice ages, limited number of cell passages, etc.
There aren&#39;t many protocols for M&#252;ller cell isolation, but there are some notable differences from this protocol to the protocols that are available5,14. First, the time between enucleation and isolation is significantly different. Other protocols suggest dissection soon after enucleation, typically within 10 min. Whereas the current protocol suggests a resting period of at least 18 h as this provided the best results for isolation in this protocol. Another significant difference between this protocol and other protocols is the point in which the eyes are treated with enzymes. In this protocol, the eyes are treated before any dissection has been performed, even before the removal of connective tissue and extraocular muscles. While other protocols isolate the retinas from the eye prior to enzyme treatment. Furthermore, the enzyme recipe is also different. In this protocol, the enzyme solution consists of trypsin and collagenase type IV. In similar protocols, papain and DNase are used for tissue digestion. Additionally, the culture vessel is different, this protocol suggests culturing on a T25 then on a T75 after the first passage. While other protocols use Petri dishes or multi-well plates. Finally, this protocol suggests that the flask should not be disturbed (for 5-7 days) to promote cell growth. Whereas in other protocols media change is performed after one day.

M&#252;ller cells (MCs) are the main and most abundant glial cells found in the retinal tissue. They are the key players in providing structural integrity and metabolic functions within the retina1. The strategic structure of MCs is spread across the entire retina thickness, thus providing support to the retina. In addition to their scaffold-like properties, they have metabolic functions for retinal neurons, supplying them with energy substrates, including glucose and lactate. These functions are crucial in order to maintain healthy neuronal function. Impaired MCs have been reported to contribute to various retinal diseases, including age-related macular degeneration, diabetic retinopathy, and glaucoma2,3. MCs can be endogenous cellular sources for regenerative therapy in the retina1. They also comprise a significant portion of the retina, and strong evidence suggests that in several species, these cells can be stimulated to substitute missing neurons2. They collaborate beneficially with neurons, and the conically branching M&#252;ller cells&#39; endfeet densely unsheathe the blood vessels and connect the retina&#39;s neural components. In order to maintain neuronal development and neuronal plasticity, M&#252;ller cells act as a soft substrate for neurons, protecting them from mechanical trauma3. Additionally, under pathological circumstances, M&#252;ller cells may differentiate into neural progenitors or stem cells that replicate or regenerate the lost photoreceptors and neurons2,3,4. M&#252;ller cells retain the characteristics of retinal stem cells, including different levels of potential for self-renewal and differentiation5,6. The M&#252;ller glial cell has a significant retinal lineage that produces neurotrophic factors, uptakes and recycles neurotransmitters, spatially buffers ions, and maintains the blood-retinal barrier in order to keep the retina in homeostasis7,8,9. This highlights the potential of M&#252;ller cells as a promising tool in cell-based therapies for treating diseases related to retinal degeneration. M&#252;ller cells are the primary glia distributed throughout the retina, connecting to both neurons and blood vessels. They play a crucial protective role, providing essential structural and metabolic support to maintain the viability and stability of retinal cells. Unfortunately, very few protocols are found in the literature for primary M&#252;ller cell isolation from the retina10,11.
We present an enhanced approach to reliably isolating and culturing mouse primary M&#252;ller cells. This protocol was used in our group to isolate M&#252;ller cells from the wild-type C57BL/6 mice and transgenic mice12,13. Mice aged between 5 and 11 days, with no sex preference, are used for this protocol. Cells have been passaged up to 4 times; however, at P4 they stop adhering to the flask, and it becomes difficult to grow a healthy culture. The culture is often contaminated with Retinal Pigmented Epithelial (RPE) cells, so the cells should be passaged at least once before performing any additional experiments on the cell line. Passaging allows for further isolation from contaminants. Therefore, the protocol presented offers a quick and effective way to isolate mouse M&#252;ller cells, which can then be used as a reliable platform to research therapeutic targets and evaluate potential treatments for retinal diseases14.

<table><tbody><tr><td>Beaker : 100mL</td><td>KIMAX</td><td>14000</td><td></td></tr><tr><td>Collagenase IV&amp;nbsp;</td><td>Worthington&amp;nbsp;</td><td>LS004188</td><td></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 (1X)&amp;nbsp;</td><td>Thermo Scientific</td><td>11885084</td><td>Media to grow M&amp;uuml;ller cells&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>gibco</td><td>26140079</td><td>For complete Muller cell culture media</td></tr><tr><td>Glutamine synthase</td><td>Cell signalling</td><td>80636</td><td></td></tr><tr><td>Heracell VISO 160i CO2 Incubator</td><td>Thermo Scientific</td><td>50144906</td><td></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>Pen Strep</td><td>gibco</td><td>15140-122</td><td>For complete M&amp;uuml;ller cell culture media</td></tr><tr><td>Phosphate Buffer saline (PBS)</td><td>Thermo Scientific</td><td>J62851.AP</td><td></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>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&quot; X 36&quot;</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>Vimentin&amp;nbsp;</td><td>invitrogen</td><td>MA5-11883</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 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.

Validation of the specificity, purity, and barrier function of isolated M&#252;ller cells 
To confirm the viability, morphology, and distinctive qualities of the isolated M&#252;ller cells, the cells were examined under a light microscope. P0 and P1 images were recorded (Figure&#160;1A). To check the contamination of isolated M&#252;ller cells with RPE cells and confirm it&#39;s purity, immunofluorescence staining (IF) was performed using antibodies specific to RPE cell marker, RPE65. Human retinal pigmented cells (ARPE -19) were used as a positive control. RPE65 stained RPE cells (red and blue for nuclear staining) are shown in Figure 1B (lower panel). RPE65 specific antibodies did not stain the isolated M&#252;ller cells (Figure 1B, upper panel). The fact that the RPE65 stained the ARPE-19 cells (red) and didn&#39;t stain the isolated cells, indicates that the isolated cells are not RPE cells. The presence of pyruvate and low glucose in the media establishes unfavorable conditions for the growth of RPE cells. Consequently, even in the event of RPE cell contamination, they will eventually detach from the flask.
Furthermore, to confirm the identity of isolated cells, a M&#252;ller cells immunofluorescence staining for M&#252;ller cells specific markers were used to confirm the successful isolation of M&#252;ller cells. The cytoskeleton intermediate filament protein called vimentin was used12,13. Additionally, glutamine synthetase (GS), that catalyzes the reaction of condensation of glutamate and ammonia to form glutamine, as a key function of retinal glial M&#252;ller cells, was also used5.
Collectively, Isolated cells stained negative for RPE65 (red, Figure 1B), positive for vimentin (green, Figure 1C) and GS (green, Figure 1D). IF staining confirms successful isolation (purity and specificity)of the isolated M&#252;ller cells. Figure 1E, is a negative control for vimentin (upper panel) and GS (lower panel), confirming the specificity of the antibodies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66237/66237fig01.jpg" /> 
Figure 1: Validation of M&#252;ller cell isolation (purity and specificity). (A)&#160;Light microscopy image for passages:passage zero(P0)and(P1)morphology. (B) RPE65 immunostaining combined with DAPI nuclear staining. (C)&#160;Vimentin immunostaining with DAPI nuclear staining at high magnification. (D)&#160;GS immunostaining at the same high magnification. (E) negative controls to confirm the specificity of vimentin and GS antibody staining. scale bar = 300 &#956;m, 300 &#956;m, 50 &#956;m, 20 &#956;m, 20 &#956;m, 50 &#956;m, 50 &#956;m, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66237/66237fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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 ...

isolation-of-primary-mouse-retinal-glial-mller-cells

Nanyang Technological University

Research

JoVE Journal

Biology

1.2K Views.  Oakland University. This manuscript describes a detailed protocol for isolating retinal glial Müller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of Müller cells.

Video: Isolation of Primary Mouse Retinal Glial Müller Cells

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

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 ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

Developmental Biology

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Neuroscience

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

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 ...

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 ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

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 ...

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

Source: Sarkar, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55364">Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility.</a> J. Vis. Exp. (2017)
The video demonstrated the method of isolating and culturing cells from a neonatal mouse brain. It involved enzymatic and mechanical dissociation of the brain, followed by culturing in growth media lacking neuronal growth factor to obtain a mixed glial population.

Uzyskiwanie mieszanej hodowli komórek glejowych z mózgu noworodka myszy

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Growing of Mixed Glial ...

Isolation of Primary Mouse Retinal Glial Müller Cells

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