Name: A Protocol for Harvesting Single-cell Suspension from Mouse Corneas (Video) | JoVE Journal
Uploaded: 2026-04-03T12:33:00.000+00:00
Duration: 4 min 50 s
Description: Learn a robust protocol for harvesting high-quality single-cell suspensions from mouse corneas using sequential enzymatic digestion. This method yields viable cells with excellent RNA integrity for single-cell RNA sequencing and flow cytometry applications.

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5 minutes

The authors would like to express their gratitude and respect to all the animals that contributed to their cells in this study. This study was supported in part by Guizhou Provincial Science and Technology Projects (QKHJC-ZK[2024]ZD043 [to S.O.]); the Fujian Provincial Science Fund for Distinguished Young Scholars (2023J06053 [to S.O]); the Natural Science Foundation of China (No.82101084 [to S.O.]; the Guizhou Provincial Basic Research Program (Natural Science)(QKHJC-ZK[2025]MS-473) [to S.O.]; the Medical Research Union Fund for High-quality health development of Guizhou Province (2024GZYKYJJKM0043) [to S.O.].

Female C57BL/6 (B6) mice, 6-8 weeks old, were supplied by the Experimental Animal Center of Guizhou Medical University. The animals were housed under a 12 h light/12 h dark cycle with ad libitum access to food and water. All procedures were approved by the Experimental Animal Ethics Committee of Guizhou Medical University (Approval No. 2305185) and conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Preparation
<ol>
	<li>Preparation of surgical instruments: Clean two pairs of ophthalmic scissors, two pairs of ophthalmic forceps, one pair of tissue forceps, and one 2 mL centrifuge tube by rinsing twice with double-distilled water (DDW). Sterilize the instruments and tube using a steam autoclave. Dry all items overnight in a conventional oven at 65 &#176;C.</li>
	<li>Solution preparation
	<ol>
		<li>Supplemental Hormonal Epithelial Medium (SHEM): Prepare SHEM by supplementing DMEM/F12 medium with the following components: 5% (v/v) fetal bovine serum (FBS), 0.5% (v/v) dimethyl sulfoxide (DMSO), 10 ng/mL mouse epidermal growth factor, 5.5 &#181;g/mL transferrin, 0.5 &#181;g/mL hydrocortisone, 1% (v/v) penicillin-streptomycin (100 U/mL penicillin, 100 &#181;g/mL streptomycin). 
		&#8203;NOTE:SHEM is used to maintain cell viability during processing. It can be substituted with alternative media such as basal DMEM or phosphate-buffered saline (PBS) containing at least 10% (v/v) FBS.</li>
		<li>Phosphate-buffered saline (PBS)
		<ol>
			<li>Dissolve 22.4 g of PBS powder in 2 L of double-distilled water (DDW) under stirring using a magnetic stirrer to obtain 1&#215; PBS.</li>
			<li>Autoclave the PBS solution one day in advance and allow it to cool.</li>
			<li>Under sterile conditions, transfer 47.5 mL of sterile 1&#215; PBS to a 50 mL centrifuge tube and add 2.5 mL of 1% (v/v) penicillin-streptomycin. 
			NOTE: The 1&#215; PBS formulation (2 L) contains 137 mM NaCl, 8 mM Na2HPO4, and 2 mM NaH2PO4, pH 7.2.</li>
		</ol>
		</li>
		<li>Collagenase A solutions
		<ol>
			<li>Stock solution: Dissolve 1 g of collagenase A powder in 40 mL of DDW. Filter sterilize through a 0.2 &#181;m filter and store at -20 &#176;C.</li>
			<li>Working solution: Dilute 1 mL of collagenase A stock solution in 4 mL of sterile DDW to obtain a final concentration of 5 mg/mL.</li>
		</ol>
		</li>
		<li>Trypsin solution: Thaw commercially available 0.25% (w/v) trypsin/EDTA solution (containing 0.91 mM EDTA) on ice before use.</li>
		<li>Trypan Blue solution (0.4%): Dissolve 0.4 g of Trypan Blue in 100 mL of DDW. Filter using filter paper and store at 4 &#176;C. 
		NOTE: The 0.4% Trypan Blue solution is used for assessing total cell count, single-cell proportion, and viability. It may be substituted with commercial live/dead staining kits or automated cell-counting systems.</li>
	</ol>
	</li>
</ol>
2. Preparation of mouse corneas
<ol>
	<li>Sterilize the dissecting microscope and work area by wiping with 75% ethanol, then expose the area to UV light inside a biological safety cabinet for 30 min.</li>
	<li>Prepare the following in sterile conditions: One 60 mm Petri dish with 5 mL of SHEM medium, three 60 mm Petri dishes each containing 5 mL of 1&#215; PBS supplemented with 5% penicillin-streptomycin (P-S), three 60 mm Petri dishes each with 5 mL of plain 1&#215; PBS, and one sterile 2 mL centrifuge tube</li>
	<li>Euthanize the mouse using CO2 asphyxiation, followed by cervical dislocation.</li>
	<li>Rinse the mouse head and closed eyelids thoroughly by immersion three times in PBS containing 5% P-S before proceeding with enucleation.</li>
	<li>Using ophthalmic scissors, make an incision along the posterior pole of each eyeball and carefully place the isolated eyes into the Petri dish containing ice-cold SHEM medium (Figure 1A). 
	NOTE: To maximize cell viability, all solutions should be pre-chilled to 4 &#176;C and all dissection steps must be performed on ice.</li>
	<li>Immediately transfer the Petri dish with eyeballs to a biological safety cabinet. Wash the eyeballs three times in 1&#215; PBS containing 5% P-S, incubating for 5 min per wash.</li>
	<li>Under a dissecting microscope, carefully separate the corneal tissue: Using ophthalmic scissors and forceps, make an incision from the posterior pole toward the corneal limbus. Gently detach the cornea from the sclera and conjunctiva along the limbal border. Remove the lens and iris completely using two pairs of forceps (Figure 1B).</li>
	<li>Using ophthalmic forceps, transfer the corneal tissue to a Petri dish containing 1&#215; PBS and wash three times (5 min each) to remove any residual non-corneal tissue and antibiotics. 
	NOTE: The iris often adheres tightly to the cornea. Ensure it is gently and completely removed using forceps to avoid contamination, leaving only clear corneal tissue.</li>
	<li>Using ophthalmic forceps, transfer the cleaned corneal tissue to a 2 mL centrifuge tube and add 1 mL of collagenase A working solution (5 mg/mL).</li>
	<li>Using ophthalmic scissors, mince the corneal tissue into approximately 0.5 mm &#215; 0.5 mm fragments directly inside the tube.</li>
	<li>Incubate the tube containing the corneal fragments and collagenase A solution in a cell culture incubator at 37 &#176;C for 1 h (Figure 2A-C).</li>
	<li>During collagenase A digestion, monitor the tissue every 10 min. Terminate digestion when no large tissue fragments remain by proceeding to centrifugation. Centrifuge the sample at 300 x g for 5 min at room temperature using a centrifuge. Carefully aspirate the supernatant.</li>
	<li>Resuspend the pellet in 200 &#181;L of 0.25% trypsin/EDTA solution.</li>
	<li>Incubate the tube in a cell culture incubator at 37 &#176;C for 10 min to digest remaining tissue fragments.</li>
</ol>
3. Isolation of corneal single cells
<ol>
	<li>Gently triturate the mixture (tissue and trypsin/EDTA solution) repeatedly using a P1000 pipette with a wide-bore tip to dissociate the tissue into a single-cell suspension.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min and discard the supernatant. Resuspend the cell pellet in 1 mL of PBS containing 0.04% bovine serum albumin (BSA) to obtain a single-cell suspension. 
	NOTE: For cells intended for sequencing or flow cytometry, resuspend in serum-free PBS. For cells that will be cultured, use 1x PBS supplemented with 0.04% BSA.</li>
	<li>Filter the cell suspension through a pre-wetted 40 &#181;m cell strainer into a new 50 mL centrifuge tube to remove cell clumps and debris. 
	NOTE: Pre-wet the cell strainer with PBS to minimize cell loss. If necessary, the filtered cells may be centrifuged again and resuspended in a desired volume for concentration.</li>
</ol>
4. Cell counting
<ol>
	<li>Combine 9 &#181;L of the cell suspension with 1 &#181;L of 0.4% Trypan Blue solution and mix gently.</li>
	<li>Slowly load the mixture into a hemocytometer chamber via the designated loading channel. Count the cells using the four-quadrant method to determine total cell number, viability, and the proportion of single cells. Perform at least two technical replicates.</li>
</ol>

<ol><li>Eghrari, A. O., Riazuddin, S. A., Gottsch, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+the+cornea%3Astructure%2C+function%2C+and+development%5BTitle%5D)+AND+%22Prog+Mol+Biol+Transl+Sci%22%5BJournal%5D)">Overview of the cornea:structure, function, and development.</a> Prog Mol Biol Transl Sci. 134, 7-23 (2015).</li>
<li>Delmonte, D. W., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomy+and+physiology+of+the+cornea%5BTitle%5D)+AND+%22J+Cataract+Refract+Surg%22%5BJournal%5D)">Anatomy and physiology of the cornea.</a> J Cataract Refract Surg. 37 (3), 588-598 (2011).</li>
<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+eye+management%3Atargeting+the+ocular+surface+microenvironment%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Dry eye management:targeting the ocular surface microenvironment.</a> Int J Mol Sci. 18 (7), 1398(2017).</li>
<li>Dou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+identity+of+human+limbal+heterogeneity+involved+in+corneal+homeostasis+and+privilege%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege.</a> Ocul Surf. 21, 206-220 (2021).</li>
<li>Adan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry%3Abasic+principles+and+applications%5BTitle%5D)+AND+%22Crit+Rev+Biotechnol%22%5BJournal%5D)">Flow cytometry:basic principles and applications.</a> Crit Rev Biotechnol. 37 (2), 163-176 (2017).</li>
<li>Hwang, B., Lee, J. H., Bang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA+sequencing+technologies+and+bioinformatics+pipelines%5BTitle%5D)+AND+%22Exp+Mol+Med%22%5BJournal%5D)">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Exp Mol Med. 50 (8), 1-14 (2018).</li>
<li>Altshuler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discrete+limbal+epithelial+stem+cell+populations+mediate+corneal+homeostasis+and+wound+healing%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing.</a> Cell Stem Cell. 28 (7), 1248-1261.e8 (2021).</li>
<li>Kaplan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA+transcriptome+helps+define+the+limbal%2Fcorneal+epithelial+stem%2Fearly+transit+amplifying+cells+and+how+autophagy+affects+this+population%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Single-cell RNA transcriptome helps define the limbal/corneal epithelial stem/early transit amplifying cells and how autophagy affects this population.</a> Invest Ophthalmol Vis Sci. 60 (10), 3570-3583 (2019).</li>
<li>Collin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+cell+atlas+of+human+cornea+that+defines+its+development%2C+limbal+progenitor+cells+and+their+interactions+with+the+immune+cells%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.</a> Ocul Surf. 21, 279-298 (2021).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+method+for+achieving+a+single-cell+suspension+from+mouse+corneas%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Optimization of method for achieving a single-cell suspension from mouse corneas.</a> Exp Eye Res. 233, 109544(2023).</li>
<li>Espana, E. M., Birk, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Composition%2C+structure+and+function+of+the+corneal+stroma%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Composition, structure and function of the corneal stroma.</a> Exp Eye Res. 198, 108137(2020).</li>
<li>Topouzi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+isolation+and+3D+culture+of+dermal+papilla+cells+from+human+hair+follicles%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles.</a> Exp Dermatol. 26 (6), 491-496 (2017).</li>
<li>Zhang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+amniotic+membrane+de-epithelialization+method+on+its+biological+properties+and+ability+to+promote+limbal+epithelial+cell+culture%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture.</a> Invest Ophthalmol Vis Sci. 54 (4), 3072-3081 (2013).</li>
<li>Hewett, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+human+endothelial+cells+from+micro-+and+macro-vessels%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and culture of human endothelial cells from micro- and macro-vessels.</a> Methods Mol Biol. 1430, 61-76 (2016).</li>
<li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Existence+of+small+slow-cycling+Langerhans+cells+in+the+limbal+basal+epithelium+that+express+ABCG2%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2.</a> Exp Eye Res. 84 (4), 626-634 (2007).</li>
<li>Song, Z., Tsai, C. H., Mei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+different+methods+to+isolate+mouse+limbal+epithelial+cells%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Comparison of different methods to isolate mouse limbal epithelial cells.</a> Exp Eye Res. 212, 108767(2021).</li>
</ol>

&#65279;The authors have no conflicts of interest to disclose.

The introduction of single-cell RNA sequencing has markedly increased the use of genomic, transcriptomic, epigenetic, and proteomic technologies in ocular research9,10,11,12. Consequently, the preparation of high-quality single-cell suspensions has become increasingly critical, as it allows the detection of signaling variations among individual cells and provides deeper insights into cellular h...

The cornea, situated at the outermost layer of the eyeball, is essential for its barrier and refractive functions, owing to a highly organized architecture comprising five distinct layers: the epithelium, Bowman&#39;s layer, stroma, Descemet&#39;s membrane, and the endothelium1. The corneal epithelium is a non-keratinized stratified squamous structure that provides a physical barrier against external pathogens and exhibits robust antimicrobial and regenerative capacities. The stroma, composed of precisely aligned collagen fibrils, maintains corneal transparency and a uniform refractive index2. The endothelium, a monocellular layer, regulates fluid transfer between the stroma and aqueous humor, thereby preserving corneal hydration and nutrient supply3. Nonetheless, this intricate structural and cellular complexity has historically posed significant challenges for investigating corneal biological functions and underlying mechanisms4.
To study intercellular interactions and identify cell-type-specific markers, techniques such as flow cytometry sorting5 and single-cell RNA sequencing (scRNA-seq)6 are widely employed. A major technical hurdle common to both approaches, however, is the preparation of high-quality single-cell suspensions. Parameters including total cell yield, proportion of single cells, and cell viability are critical for the reliability of downstream applications aimed at characterizing corneal cell function and surface marker expression7. Despite its importance, there remains a lack of standardized and efficient protocols for generating single-cell suspensions from whole mouse corneas.
Several methods have been established for obtaining single-cell suspensions from corneal tissue. Sequential enzymatic digestion using collagenase A, trypsin, or TrypLE has been employed to isolate single cells from both human and murine corneas8. Repeated trypsinization has also been utilized for corneal dissociation&#8311;. Additionally, commercial multi-tissue dissociation kits, such as those from Miltenyi Biotec, have been successfully applied to generate corneal single-cell suspensions9. In our previous comparative study of different corneal digestion methods, we demonstrated that sequential treatment with collagenase and trypsin constitutes the optimal approach. This method consistently yields high cell numbers, excellent viability (&#8805;94.6%), a high percentage of single cells (96.3%), and well-preserved RNA integrity10.
In this protocol, we provide a detailed, step-by-step procedure for generating high-quality single-cell suspensions from whole mouse corneas using this optimized enzymatic digestion strategy, based on our established experience and prior findings.

<table><tbody><tr><td>Alexa Fluor 594 donkey anti-rabbit</td><td>Abcam</td><td>ab150064</td><td></td></tr><tr><td>Antikeratin</td><td>Abcam</td><td>ab230175</td><td></td></tr><tr><td>Anti-pancytokeratin</td><td>Thermo Fisher Scientific</td><td>AE1/AE3, Alexa FluorTM 488</td><td></td></tr><tr><td>Autoclave sterilizer</td><td>An Shen</td><td>LDZH-100KBS</td><td></td></tr><tr><td>Bovine Serum Albumin V</td><td>Solarbio</td><td>A8020</td><td></td></tr><tr><td>Cell filter</td><td>NEST</td><td>258369</td><td>40 &amp;mu;m</td></tr><tr><td>Centrifugal machine</td><td>Eppendorf AG</td><td>22331 Hamburg</td><td></td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td></td></tr><tr><td>Confocal microscope</td><td>&amp;nbsp;Oberkochen</td><td>Zeiss LSM 880</td><td></td></tr><tr><td>Counting plate</td><td>MARIENFELD</td><td>M407859</td><td></td></tr><tr><td>Centrifugal tube</td><td>NEST</td><td>620611</td><td>2 mL, 50mL</td></tr><tr><td>Coverslip</td><td>MARIENFELD</td><td>M407859</td><td></td></tr><tr><td>DAPI</td><td>Solarbio</td><td>C0065</td><td></td></tr><tr><td>Dissecting microscope</td><td>Carl ZEISS</td><td>VISU150</td><td></td></tr><tr><td>DMEM/F12&amp;nbsp; media</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>DMSO</td><td>Solarbio</td><td>D8371</td><td></td></tr><tr><td>Fetal&amp;nbsp; bovine&amp;nbsp; serum</td><td>Gibco</td><td>16140071</td><td></td></tr><tr><td>Hydrocortisone</td><td>Solarbio</td><td>IH0100</td><td></td></tr><tr><td>ITS</td><td>Gibco</td><td>23337619</td><td></td></tr><tr><td>Mouse epidermal growth factor</td><td>Invitrogen</td><td>PMG8041</td><td></td></tr><tr><td>ophthalmic surgical&amp;nbsp; forceps</td><td>Dumot</td><td>0208-5-PO</td><td></td></tr><tr><td>ophthalmic surgical scissors</td><td>FST</td><td>15021-15</td><td></td></tr><tr><td>Oven</td><td>AISITE</td><td>DH6000 II</td><td></td></tr><tr><td>Penicillin &amp;amp;Streptomycin</td><td>Gibco</td><td>15070063</td><td>100mL</td></tr><tr><td>petri dish</td><td>NEST</td><td>705201</td><td>60mm</td></tr><tr><td>Phosphate buffered saline</td><td>Solarbio</td><td>P1010</td><td></td></tr><tr><td>Thermostatic incubator</td><td>Thermo Fisher Scientific</td><td>3111</td><td></td></tr><tr><td>Triton-X 100</td><td>Solarbio</td><td>T8200</td><td></td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Solarbio</td><td>T8070</td><td></td></tr><tr><td>Trypsin/EDTA&amp;nbsp;</td><td>Gibco</td><td>R001100</td><td></td></tr><tr><td>Ultra-cleaning table</td><td>You Qi</td><td>VS-840-2</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Solarbio</td><td>P1110</td><td></td></tr></tbody></table>

a protocol for harvesting single-cell suspension from mouse corneas

A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

As the outermost layer of the eyeball, the cornea plays a critical role in maintaining ocular surface homeostasis and vision. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled high-resolution analysis of corneal cellular heterogeneity, yet the preparation of high-quality single-cell suspensions from corneal tissue remains challenging due to its dense and complex structure. In this study, we establish a robust protocol for generating single-cell suspensions from whole mouse corneas. The procedure involves mincing the corneas into fine fragments followed by sequential enzymatic digestion using collagenase A and trypsin. The resulting cell suspension was assessed for quality through multiple metrics: trypan blue staining was used to evaluate cell viability and count; immunofluorescence staining for Pancytokeratin and Keratocan enabled identification and quantification of major corneal cell types; and RNA integrity was verified using a bioanalyzer. This protocol consistently yields single-cell preparations with high cell numbers, excellent viability (&#8805; 94.6%), and a high proportion of single cells (96.3%), fulfilling the requirements compatible with demanding downstream applications, including scRNA-seq.

The quality of the single-cell suspensions was assessed based on total cell yield, proportion of single cells, and cell viability. The suspension was stained with Trypan Blue and analyzed using phase-contrast microscopy across multiple fields (Figure 3). From four mouse eyes (eight corneas), a total of 1.05 × 105 cells were obtained, with an average cell viability of 94.6% and a single-cell ratio exceeding 96.3%. These results confirm that the prepared cell suspension is suitable ...

Here, we present a protocol to obtain high-quality single-cell suspensions from whole mouse corneas through sequential digestion with collagenase A and trypsin. This method yields viable single cells with high RNA integrity, making them compatible with downstream applications such as single-cell RNA sequencing and flow cytometry.

As the outermost layer of the eyeball, the cornea plays a critical role in maintaining ocular surface homeostasis and vision. Recent advances in ...

This protocol primarily introduces a method for obtaining high quality single cells from intact mouse corneas. Our method is viable single cells with high RNA integrity, making them compatible with downstream applications such as single cell on a thick thin and flow diametry. To begin, position a six to eight-week-old euthanized female C57BL6 mouse on a sterile dissection table.After rinsing the closed eyelids with PBS, use ophthalmic scissors to make an incision along the posterior pole of the eyeball, and carefully place the isolated eye into a Petri dish containing ice cold supplemental hormonal epithelial medium. After rinsing the eyeball three times with 1XPBS supplemented with 5%penicillin streptomycin, use ophthalmic scissors and forceps to make an incision from the posterior pole toward the corneal limbus. Gently detach the cornea from the sclera and conjunctiva along the limbal border, then using two pairs of forceps, completely remove the lens and iris.After washing the corneal tissue three times in PBS, transfer the clean tissue to a new tube. Add one milliliter of five milligrams per milliliter collagenase A solution, and mince the tissue into fragments of approximately 0.5 by 0.5 millimeters using ophthalmic scissors. Incubate the tube containing the corneal fragments with collagenase A solution in a cell culture incubator at 37 degrees Celsius for one hour.Inspect the tissue every 10 minutes during the digestion. When no large tissue fragments remain, centrifuge the sample at 300G for five minutes at room temperature, then carefully aspirate the supernatant. Next resuspend the pellet in 200 microliters of 0.25%trypsin EDTA solution.Place the tube in a cell culture incubator set to 37 degrees Celsius for 10 minutes to digest remaining tissue fragments. Finally, use a P1000 pipette fitted with a wide bore tip to gently triturate the mixture to obtain a single cell suspension. Centrifuge the cell suspension as demonstrated earlier, and resuspend the cell pellet in PBS containing 0.04%BSA.Upon counting the isolated corneal cells using a hemocytometer, it was found that a total of 1.05 times 10 to the power of five cells were obtained from eight corneas with an average cell viability of 94.6%and a single cell ratio exceeding 96.3%confirming the suspension suitability for downstream applications. Immunofluorescence analysis showed that the cell suspension comprised 83.5%pan-CK-positive epithelial cells, 13.5%keratocan positive stromal cells, and approximately 3%cells negative for both markers. The RNA extracted from the single cell suspension exhibited a high RNA integrity number of around 8.8 and a 28S by 18S ribosomal RNA ratio of about 2.15, confirming excellent RNA quality.After obtaining single cells, we can characterize corneal cell function and surface mark exploration. This vicinity the study of cell-to-cell interactions. A challenge in this method is virtually separating the cornea from other tissues.

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The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

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.

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Learn a robust protocol for harvesting high-quality single-cell suspensions from mouse corneas using sequential enzymatic digestion. This method yields viable cells with excellent RNA integrity for single-cell RNA sequencing and flow cytometry applications.

A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

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