isolating murine retinal ganglion cells using fluorescence-activated cell sorting

Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

Place up to 12 retinae onto a sterile 70-micron nylon strainer moistened with PBS and FBS, and gently macerate the retina with the back end of a 10-milliliter syringe plunger using a circular motion. When all of the cells have been dissociated, place the strainer over a polypropylene collection tube, and use a P1000 pipette to filter the cells through the strainer into the collection tube.
Rinse the strainer with PBS and FBS, pooling the wash in the collection tube. Add enough PBS plus FBS to bring the final volume up to 1 milliliter of solution per retina, and collect the cells by centrifugation. Then, resuspend the pellet in PBS plus FBS at a 1 milliliter of medium per 5 retinae concentration. Next, block any nonspecific FC receptor binding in each tube with 1 microliter of anti-mouse CD16/32 antibody per 1 x 106 cells for 10 minutes at room temperature.
At the end of the blocking incubation, add the antibody cocktail of interest to the cells with gentle pipetting for a 30-minute incubation on ice protected from light. Then, wash the cells two times in a 4-milliliter final volume of PBS and FBS. Label the cells with an appropriate secondary antibody for another 30 minutes on ice protected from light, followed by 2 washes in PBS plus FBS, and load the sample tube onto the flow cytometer.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Instruments, Solutions, and Media
Note: All information about materials, reagents, tools, and instruments reported in the protocol are specified in the Table of Materials.
<ol>
	<li>Autoclave all dissection instruments and store them in a sterile area. Use the following instruments: 4 standard forceps (2 long and 2 short) and 2 scissors, as well as 2 forceps (1 long and 1 short) and 1 scissor for the dissection; keep an extra set as a backup.</li>
	<li>Prepare 100 mL of sterile phosphate-buffered saline (PBS)/1% fetal bovine serum (FBS) solution to use during washes, immunolabeling procedures, and cell sorting steps. Keep the solution chilled at 4 &#176;C. 
	Note: Do not add sodium azide (NaN3) to the solution, as it can be toxic to live cells.
	<ol>
		<li>Prepare 100 mL of PBS/1% FBS with 99 mL of PBS and 1 mL of FBS.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of sterile neural cell medium supplemented with 3% FBS (see Table of Materials) for use as collection and culture medium. Keep cell culture medium sterile at 4 &#176;C. Only warm it to room temperature (RT) prior to use.
	<ol>
		<li>Prepare 100 mL of neural cell medium supplemented with 3% FBS using 97 mL of neural cell medium and 3 mL of FBS.</li>
	</ol>
	</li>
	<li>Pre-chill collection tubes (15-mL tubes) pre-coated with 5 mL of collection medium by placing them in an ice bucket. Only use polypropylene tubes to prevent the cells from adhering to the tube surface.</li>
	<li>Place 40-mm dishes, 70-&#181;m nylon strainers, syringes, pestles for the cell strainer, and sterile polypropylene tubes in the biosafety cabinet. Sterilize all items prior to the procedures and maintain them in a sterile manner throughout the procedures. 
	Note: All steps after the collection of the retinae will be performed in the biosafety cabinet.</li>
</ol>
2. Preparation of the Retinal Cell Suspension
<ol>
	<li>Place the collected eye on the base plate of a dissection microscope to begin the corneal dissection. Dissect each eye individually.</li>
	<li>Carefully hold the globe at the optic nerve base using forceps. For this step, use one long and one short standard forceps.</li>
	<li>Use a 30-gauge (30G) sharp needle to puncture the cornea. This will allow the aqueous humor to evacuate from the eye, making it easier to hold the eye with the forceps.</li>
	<li>Hold the cornea with the forceps and use scissors to make a small incision in the cornea. Gently peel the cornea and sclera using the forceps. When the globe is peeled halfway, roll the retina and lens out using the forceps. Discard the cornea, sclera, and lens. 
	Note: This method ensures that the retina completely detaches from the remainder of the eye.</li>
	<li>Place the retina in a small 40-mm Petri dish containing PBS/1% FBS. 
	Note: As an alternative to the Petri dish, a cell culture dish can be used. Make sure to always keep the retinae moist, as in physiological conditions.
	<ol>
		<li>Wash each retina three times with fresh sterile PBS/1% FBS within the biosafety cabinet.</li>
	</ol>
	</li>
	<li>Place up to 12 retinae on a sterile 70-&#181;m nylon strainer moistened with PBS/1% FBS. Using the back end of a 10-mL syringe, gently macerate the retinae using a circular movement to detach the cells.
	<ol>
		<li>Alternatively, use a pestle for the cell strainer maceration or use enzymatic digestion with a combination of 15 IU/mL papain, 5 mM L-cysteine, and 200 U/mL DNase I for 15 min at 37 &#176;C, followed by inactivation with PBS/10% FBS. 
		Note: No changes in the percentage of cells recovered were observed when enzymatic digestion was compared to maceration with either the back end of the syringe or the pestle.</li>
	</ol>
	</li>
	<li>To transfer the isolated cells, place the sterile 70-&#181;m nylon strainer over the polypropylene collection tube. Pass the collected cells through the strainer using a P1000 pipette. Rinse the strainer (step 3.6) with PBS/1% FBS to release any remaining cells and transfer them to the collection tube.</li>
	<li>Add PBS/1% FBS to achieve a final volume of 1 mL per retina. Centrifuge the cell suspension for 7 min at 200 x g and RT. 
	&#8203;Note: Depending on the number of macerated retinae (&#60; 6 retinae), the cell pellet may be too small to be visible.
	<ol>
		<li>Discard the cell supernatant and resuspend the cell pellet in PBS/1% FBS using a ratio of 1 mL per 5 retinae.</li>
	</ol>
	</li>
	<li>Count the cells using a hemocytometer.
	<ol>
		<li>Clean a glass hemocytometer and coverslip with 70% alcohol. Gently swirl the tube containing the cells to ensure that the retina cell suspension is evenly distributed. Place 20 &#181;L of 0.4% trypan blue in a microcentrifuge tube and mix with 20 &#181;L of the cell suspension.</li>
		<li>Mix gently and apply 10 &#181;L of the 0.4% trypan blue/cell suspension mix to the hemocytometer, filling both chambers; capillary action will draw the 0.4% trypan blue/cell suspension mix under the coverslip. Using a microscope, count all trypan blue-negative cells; this number represents the live cells.</li>
		<li>Determine cell viability using the following formula: live cell count / total cell count = % viability. 
		Note: If the viability of the cells is less than 95%, a fluorescent dye is required to discriminate cell viability during fluorescence-activated cell sorting (FACS).</li>
	</ol>
	</li>
	<li>Use a fluorescent viability dye that is non-permeant to live cells. Wash the cells with PBS to remove any trace of serum. Add 1 &#181;L of fluorescent dye for cell viability discrimination per 5.0 x 106 cells in a 100-&#181;L final volume. Incubate at RT for 15 min, protected from light. Add 10x the volume of PBS/1% FBS. Centrifuge for 5 min at 200 x g and RT.</li>
	<li>If necessary, incubate the cell suspension overnight at 4 &#176;C. If this is done, fill the cell suspension tube with neural cell medium rather than PBS/1% FBS. Place the tube horizontally and keep it at 4 &#176;C.</li>
</ol>
3. Immunolabeling the Retinal Cells
<ol>
	<li>Use the following conversion to determine the volume of antibody per cell number: 2 &#181;L of antibody per 5.0 x 106 cells in a 100-&#181;L volume.</li>
	<li>Wash the cells and maintain them in PBS/1% FBS. Take a small aliquot of cells (5.0 x 106 cells) to use as a negative control (unlabeled) at the time of the sort set up. 
	Note: This negative control is critical for the proper calibration of the cell sorter.</li>
	<li>To minimize the non-specific binding of antibodies to cells that express the Fc&#947; receptors II and III, add 1 &#181;L of an anti-mouse CD16/32 antibody per 1.0 x 106 cells in a 50-&#181;L final volume using PBS/1% FBS. Incubate for 10 min at RT.</li>
	<li>Add the antibody cocktail to the sample and mix gently by pipetting. Incubate for 30 min in an ice bucket. Ensure that the ice bucket is covered, as light could compromise the experiment due to the photobleaching of the tagged fluorophores.
	<ol>
		<li>Prepare the antibody cocktail using the following fluorescently tagged anti-mouse antibodies: CD90.2 AF (Alexa fluor)-700, CD48 PE (Phycoerythrin)-Cyanine7, CD15 PE, and un-tagged CD57. 
		&#8203;Note: For each antibody, use the following concentrations per 5.0 x 106 cells: CD90.2 AF700, 1 &#181;g; CD48 PE-Cyanine7, 0.4 &#181;g; CD15 PE, 0.02 &#181;g; and un-tagged CD57, 0.4 &#181;g.
		<ol>
			<li>Use volumes as per the following calculations (based on the commercially available antibodies listed in the Table of Materials): total of 5.0 x 107 cells to be labeled; 2 &#181;L per 5.0 x 106 cells = 20 &#181;L of each antibody; 4 different antibodies = 80 &#181;L of antibody cocktail; final volume = [(5.0 x 107 total cells)/5.0 x 106 cells] x 100 = 1,000 &#181;L; 80 &#181;L Abs + 50 &#181;L of anti-mouse CD16/32 + 870 &#181;L PBS/1% FBS = 1,000 &#181;L. 
			Note: This combination provided the optimal combination of fluorochromes for the instrument configuration. The following parameters summarize the emission and excitation: AF-700, emission of 719 nm when excited with a 638-nm red diode laser; PE-Cyanine7, emission of 767 nm when excited with a 488-nm blue diode laser; and PE, emission of 575 nm when excited with a 488-nm blue diode laser.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After the 30-min incubation, bring volume up to 5 mL using PBS/1% FBS. Wash the samples by centrifuging them for 7 min at 200 x g and RT. Repeat the procedure once to remove all unbound antibodies.</li>
	<li>Add 2 &#181;L of secondary antibody (0.1 &#181;g), which will bind the CD57 of 5.0 x 106 cells. Incubate for 30 minutes in an ice bucket, as in step 3.4. Wash the cells twice, as in step 3.5.
	<ol>
		<li>Label the cells with a secondary antibody if an un-tagged primary antibody was used in step 3.4. 
		&#8203;Note: The secondary antibody of choice for this configuration is listed in the Table of Materials; it has an emission wavelength of 421 nm. Use it with a bandpass filter of 450/50 nm when excited with the violet diode laser at 405 nm.</li>
		<li>Use volumes as per the following calculations (based on the commercially available antibody listed in the Table of Materials): 2 &#181;L per 5.0 x 106 cells = 20 &#181;L of secondary antibody; final volume = [(5.0 x 107 total cells)/5.0 x 106 cells] x 50 = 500 &#181;L; 20 &#181;L of Abs + 480 &#181;L PBS/1% FBS = 500 &#181;L.</li>
	</ol>
	</li>
	<li>Keep the labeled cells in PBS/1% FBS. Count the cells using a hemocytometer; use a final retinal cell concentration of 3.0 - 4.0 x 107 cells/mL. 
	Note: Do not use cell culture medium to dilute the cells because the phenol red can increase the autofluorescence, thereby reducing the resolution between negative and positive cells. The final cell concentration per volume highly depends upon the volume flow rate of the instrument configuration.</li>
	<li>Use single-color controls during setup to minimize fluorescence spillover. 
	Note: This step, also known as compensation, is applied to correct the noise created by the combination of fluorophores. Polystyrene microspheres in PBS/0.1% BSA/2 mM NaN3 with the capacity to bind fluorescently tagged immunoglobulin (Ig) isotypes from multiple species provide positive controls to set the compensation.
	<ol>
		<li>Place 3 drops of the polystyrene microspheres into sterile FACS tubes, allotting one per fluorophore. Add 1 &#181;g of the respective fluorophore to each tube. Incubate for 15 min at RT, protected from light. Add 3 mL of PBS/1% FBS to the sample tubes. Centrifuge for 5 min at 200 x g and RT. Carefully remove the supernatant and resuspend in 250 &#181;L of PBS/1% FBS.</li>
		<li>To set up the negative controls, use polystyrene microspheres that do not bind to Ig. If necessary, carry out this step the day before.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse CD15 PE</td>
			<td>BioLegend</td>
			<td>125606</td>
			<td>Clone MC-480</td>
		</tr>
		<tr>
			<td>Anti-mouse CD48 PE-Cy7</td>
			<td>BioLegend</td>
			<td>103424</td>
			<td>Clone HM48-1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD57</td>
			<td>Sigma Aldrich</td>
			<td>C6680-100TST</td>
			<td>Clone VC1.1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD90.2 AF700</td>
			<td>BioLegend</td>
			<td>105320</td>
			<td>Clone 30-H12</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 Goat Anti-mouse IgG</td>
			<td>BioLegend</td>
			<td>405317</td>
			<td>Clone Poly4053</td>
		</tr>
		<tr>
			<td>Purified Anti-mouse CD16/32</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>FcgRII/III block, Clone 93</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>U.S. origin</td>
		</tr>
		<tr>
			<td>AbC Total Antibody Compensation Bead Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10497</td>
			<td>Multi-species Ig</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Add serum to media prior to culture.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>Saline solution</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Olympus</td>
			<td>SZ-PT Model</td>
			<td>Stereo Microscope</td>
		</tr>
		<tr>
			<td>Sorvall Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>ST 16R</td>
			<td>All centrifugation performed at RT</td>
		</tr>
		<tr>
			<td>Base Plate &#8211; Dissection Pan</td>
			<td>Fisher Scientific</td>
			<td>SB15233FIM</td>
			<td>A wax plate can also be used</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Aesculap</td>
			<td>5002-7</td>
			<td>4 &#189; inches</td>
		</tr>
		<tr>
			<td>Iris Scissors, Straight</td>
			<td>Aesculap</td>
			<td>1360</td>
			<td>5 &#189; inches</td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>BD FACS Tubes</td>
			<td>Fisher Scientific</td>
			<td>352003</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>40 mm dishes</td>
			<td>MidSci</td>
			<td>TP93040</td>
			<td>Tissue culture treated</td>
		</tr>
		<tr>
			<td>70 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>70ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>40 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>40ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>BD 10 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>301604</td>
			<td>Disposable Syringe without needle</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>MidSci</td>
			<td>PEST</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Wheaton Vials</td>
			<td>Fisher Scientific</td>
			<td>986734</td>
			<td>No Liner</td>
		</tr>
		<tr>
			<td>BD 30 G needle</td>
			<td>Fisher Scientific</td>
			<td>305128</td>
			<td>1 inch</td>
		</tr>
		<tr>
			<td>Hausser Scientific Bright-Line Glass Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>0267151B</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>Gibco Trypan blue 0.4% Solution</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td>Viability Dye</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>05-402-25</td>
			<td>1.5mL</td>
		</tr>
		<tr>
			<td>EVOS Floid Cell Imaging</td>
			<td>Thermo Fisher Scientific</td>
			<td>447113</td>
			<td>Fluorescence Imaging with a 20x objective</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-452</td>
			<td>Used to make 70% Ethanol</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014282</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Rack LTS 1000 mL &#8211; GPS-L1000S</td>
			<td>Rainin</td>
			<td>17005088</td>
			<td>Blue Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 250 mL &#8211; GPS-L250S</td>
			<td>Rainin</td>
			<td>17005092</td>
			<td>Green Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 20 mL &#8211; GPS-L10S</td>
			<td>Rainin</td>
			<td>17005090</td>
			<td>Red Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>FACSAria II Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>LSR II Cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
	</tbody>
</table>

Source: Chintalapudi, S. R., et al. <a href="https://app.jove.com/v/55785">Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure for isolating murine primary retinal ganglion cells (RGCs) by using flow cytometry. This method allows 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.

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

Place a murine retina on a moistened cell strainer and macerate in a circular motion to separate the retinal cells from the extracellular matrix.
Transfer the strainer to a collection tube.
Add a phosphate buffer and serum solution to the strainer to wash and collect the cells.
Centrifuge to settle the cells. Discard the supernatant and resuspend the cells in a phosphate buffer and serum solution.
Add blocking antibodies that bind to Fc receptors on cells, preventing non-specific binding of target antibodies.
Next, add fluorescently tagged and untagged primary antibodies that bind to specific cell surface markers on various cell types.
Wash to remove unbound antibodies.
Add a fluorescently tagged secondary antibody to bind to the untagged primary antibody.
Wash to remove unbound antibodies.
Perform fluorescence-activated cell sorting, or FACS to capture the distinct fluorescent signals of the labeled cells and sort the retinal ganglion cells from various retinal cell populations.

18 Views. בידוד תאי גנגליון רשתית מורין באמצעות מיון תאים המופעלים על ידי פלואורסצנטיות

Video: בידוד תאי גנגליון רשתית מורין באמצעות מיון תאים המופעלים על ידי פלואורסצנטיות - Experiment

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

יחיד תא RNA-Seq של קבוצות מוגדרות של תאים גנגליון רשתית

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

JoVE Journal

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Coculture של נוירונים רשתית עכברוש אקסוטומי עם חוש הריח Ensheathing גליה, כמודל במבחנה של התחדשות אקסון למבוגרים

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

בידוד רשתית עכבר וטיפול במתנול לחקר תאי גנגליון ברשתית

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

מחבר זרקור: הכנה מלאה מבוססת מתנול לחקר תאי גנגליון ברשתית  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

תכשיר שלם על בסיס מתנול לחקר תאי גנגליון ברשתית

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

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Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting