Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia–derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3–6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

Adult mammals lack the ability to regenerate retinal neurons after injury.However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina.Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist.However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed.As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells.In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987.

Background
Adult mammals cannot typically regenerate retinal neurons after injury [1][2][3].However, previous research from this lab using adult mice has shown that the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, can induce neurogenesis in adult mammals when applied as eyedrops in the presence or absence of any retinal injury [4][5][6][7][8][9][10].PNU-282987 is believed to act on alpha7 nAChRs in the retinal pigment epithelium to release signaling molecules onto the end feet of Müller glia to introduce cell cycle re-entry.From there, Müller glia dedifferentiate and form retinal progenitor cells that eventually develop into mature retinal neurons [6,9].However, these studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus restricting the number of antibodies that can be multiplexed and limiting the examination of cells undergoing mitosis and proliferation.As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells.In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with mitotically active and proliferating cells that arise after treatment with PNU-282987.

B. Retina harvesting
1.After euthanasia by carbon dioxide asphyxiation, place Dumont #7 curved forceps around the posterior aspect of the eyeball near the optic nerve.Apply gentle pressure and lift the eyeball out of the cavity.Place in a 10 mm Petri dish filled with 3 mL of ice-cold 1× PBS (Figure 1A). 2. Grasping the sclera with Dumont #5 forceps, move the eye out of the Petri dish.Create a puncture hole using a scalpel along the corneal equator (Figure 1B and 1C). 3. Hold the edge of the puncture hole with Dumont #5 forceps and place the eye back into the Petri dish.
Insert Spring scissors into the puncture hole to create a circumferential incision along the corneal equator (Figure 1D). 4. Using a second pair of Dumont #5 forceps, begin to peel away the cornea and the iris from the posterior eye cup.Grasping the optic nerve with Dumont #5 forceps to generate traction may be necessary at this step (Figure 1E and 1F). 5. Once the cornea and iris are peeled away, the lens will be fully revealed.Remove the lens from the posterior eye cup with Dumont #5 forceps (Figure 1G).6. Evert the posterior eyecup with Dumont #5 forceps to prolapse the retina (Figure 1H).

C. Retina dissociation
1. Follow the manufacturer's instructions for preparing papain, DNase I, and ovomucoid inhibitor to yield the proper concentrations.2. Transfer the four retinae using Dumont #5 forceps into a 1.5 mL microcentrifuge tube filled with 500 μL of papain that has been warmed to 37 ℃ for at least 10 min in a water bath.3. Mince the retinae into eight pieces using Spring scissors.4. Add 25 μL of 1 mg/mL DNase I to papain. 5. Close the microcentrifuge tube lid and place it in an incubator heated to 37 ℃ on a rocker for constant agitation for 60 min.Note: Optimal papain incubation time was determined in a separate experiment to investigate which incubation time yielded the highest number of living cells (Figure S1). 6. Pour the solution into a 90 mm Petri dish.Use warm EBSS to rinse out any remaining retinal tissue.7. Triturate the tissue with a 10 mL pipette at least five times or until the solution appears cloudy and there are no large pieces of retinal tissue remaining.Transfer contents to a 15 mL conical tube.

Caution:
The introduction of bubbles during trituration should be avoided.Bubbles have a high surface tension that can cause lysis.8. Centrifuge at 300× g for 5 min.Caution: It is important to mixing the two-phase solution.We have found that the best way to avoid mixing is to tilt the Pipet-Aid containing the cell suspension horizontally and release the liquid on the "slow" setting.12. Centrifuge at 70× g for 6 min.
Note: A flow cytometry plot depicting a freshly dissociated retina is represented in Figure 2.

D. Fixation and permeabilization
1. Discard supernatant and resuspend the cell pellet by triturating it 3-5 times in 2 mL of 4% PFA using a 10 mL pipette.Let this sit for 15 min at room temperature for fixation.2. Centrifuge at 500× g for 5 min.3. Discard supernatant in hazardous waste and wash cell pellet by resuspension, triturating it 1-2 times in 2 mL of FACS buffer.4. Centrifuge at 500× g for 5 min.5. Discard the supernatant and resuspend the cell pellet by triturating it 3-5 times in 2 mL of 0.1 M HCl that has been prewarmed to 37 ℃ using a 10 mL pipette.Incubate at 37 ℃ in a warmer for 15 min.6. Centrifuge at 500× g for 5 min.7. Discard the supernatant and resuspend the cell pellet by triturating it 3-5 times in 2 mL of 0.4% Triton-X 100 using a 10 mL pipette.Let this sit for 15 min at room temperature for permeabilization.Note: A flow cytometry plot depicting a fixed and permeabilized retina is represented in Figure 3. 8.While incubating, collect 20 μL of the suspension and count cells using an automated cell counter or manually using a hemocytometer.Note: Cell counts are approximately 1 × 10 6 per two retinas.9. Centrifuge at 500× g for 5 min.10.Remove the supernatant.Add 20 μL of Fc block and 50 μL of BD Horizon Brillant Stain Buffer to all experimental tubes.11.Incubate for 15 min at room temperature.12. Add 2 mL of FACS buffer using a 10 mL pipette and centrifuge at 500× g for 5 min.
Discard the supernatant and resuspend cells in 1 mL of FACS buffer by triturating it 1-2 times using a 1 mL glass pipette.Remove suspension from 15 mL conical using a 1 mL glass pipette and add it to a 1.5 mL microcentrifuge tube.The following groups should be created: A) single-stain compensation beads for each color in separate 1.5 mL microcentrifuge tubes (Table 2); B) untreated/unstained cells (Table 3); C) DMSO control (Table 3); and D) PNU-282987 treated ( Compensation controls must be repeated for every experiment.In the case of the retina, we found beads are the most appropriate material for compensation as compared to retina cells.b.Please note that FMOs are utilized to determine positive vs. negative gates for the fluorophores included in the panel.FMOs need to be run during panel setup.An example of the FMO panels used in this experiment can be found in Figure S2.

General notes and troubleshooting
The discontinuous density gradient where the two layers of the gradient are visible, created in the dissociation protocol, is essential for a high cell yield.We have found that the best way to create a proper gradient is to turn the Pipet-Aid speed setting to slow, hold the Pipet-Aid horizontally so that the opening of the serological pipette is touching the inside of the tube, and slowly release the suspension so that it delicately layers.Gentle or overly vigorous trituration of retinal tissue after incubation with papain during the dissociation process will result in a low yield of cells.Additionally, using a pipette that has a small opening could shear the cells.Therefore, we have found the tissue must be triturated at least five times with a 10 mL serological pipette so that the resulting solution is cloudy.However, if the resulting cell numbers are low with five triturations, lowering the number of triturations could result in a higher yield.

Figure 1 .
Figure 1.Illustration demonstrating retina dissection.(A) Submerge the enucleated mouse eye in icecold 1× PBS in a 10 mm Petri dish.(B) Using Dumont #5 forceps, grasp the sclera and move the eye outside of Petri dish.While securing the eye in place with Dumont #5 forceps, create a scleral puncture using a scalpel along the corneal equator.(C) The black rectangle surrounded by the dotted circle depicts where the puncture wound should be placed.(D) Using Dumont #5 forceps, grasp the edge of the puncture wound to place the eye back into the dish.Insert Spring scissors into the puncture wound and create a circumferential incision along the corneal equator.(E) Using a second pair of Dumont #5 forceps, start peeling away the cornea and the iris from the posterior eye cup.(F) Grasp the optic nerve to generate traction.(G) Remove the lens while grasping the posterior eyecup with Dumont #5 forceps.(H) Prolapse the retina by everting the posterior eyecup with Dumont #5 forceps.(I) Secure the posterior eyecup in place with Dumont #5 forceps.Detach the retina from the posterior eyecup with Spring scissors.If any black pigmented retinal pigment epithelium remains, gently remove it with Spring scissors.(J) View depicting the isolated retina after dissection.After dissection, the isolated retina should be rinsed by submerging it in a new Petri dish filled with ice-cold 1× PBS.

7 Published 9 .
Cite as: Vanzo-Sparks, H.K. et al. (2024).Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.Bio-protocol 14(13): e5024.DOI: 10.21769/BioProtoc.5024.While contents are centrifuging, create a resuspension solution in a 15 mL conical tube containing 2.7 mL of EBSS, 300 μL of ovomucoid inhibitor solution, and 150 μL of DNase I. 10.Remove the supernatant.Using a 10 mL pipette, transfer the entire resuspension solution created in step C9 to the conical tube containing the cell pellet and resuspend the cell pellet by triturating it 1-2 times.11.Prepare discontinuous density gradient.In a new 15 mL conical tube, transfer 5 mL of ovomucoid inhibitor solution prepared in step C1 using a 10 mL pipette.Next, using a 10 mL pipette, add the entirety of the cell suspension created in step C10 on top of the 5 mL of albumin-ovomucoid solution to create a twophase solution.

Figure 2 .
Figure 2. Unfixed and unpermeabilized dissociated retinae.(A) Plot depicting unfixed and unpermeabilized dissociated retinae with no fluorescent antibodies added.(B) Parameters include FSC (forward scatter; size) and SSC (side scatter; granularity).The corresponding voltages were implemented to detect an optimal signal above the noise and resolve positive and negative populations.

Figure 4 .
Figure 4. Representative gating strategy used for gating vimentin+ and Ki67+ cells after 28 days of DMSO and PNU-282987 treatment.(A) and (F) A gate is made around the main cell population to exclude debris (SSC-A vs. FSC-A).(B-C) and (G-H) Singlet gates are drawn using FSC-H vs. FSC-A and SSC-H vs. SSC-A.(D) and (I) Vimentin+ cells are gated using FMOs to determine the cell population positive for vimentin (FSC-A vs. BUV-395-A).Back-gating figure for vimentin, displaying vimentin+ cells in the parent populations, is depicted in Figure S3.(E) and (J) Vimentin+ cells that are also labeled for the proliferation marker Ki67 are gated around.FMOs were utilized to determine positive cell populations (BUV-737-A vs. BUV-395-A).

Figure 5 .
Figure 5. Müller glia and proliferating Müller glia are increased in PNU-282987-treated samples.Adult (3-6 month old) mice were treated with 1× PBS eyedrops containing 1% DMSO and 1 mg/mL BrdU or 1 mM PNU-282987 and 1 mg/mL BrdU for 28 days.Subsequently, retinae were dissociated and processed for analysis by flow cytometry.(A) Graph showing the percent increase of vimentin+ Müller glia cells in DMSO vs. PNU-282987treated samples.(B) Graph showing the percent increase of vimentin+Ki67+ Müller glia in DMSO vs. PNU-282987treated samples.Total retinae: DMSO n = 12; PNU-282987 n = 12.Results are based on three independent experiments with retinae from mice pooled in each of the three experiments.Statistics: unpaired t-test with Welch's correction; *p < 0.05.