Materials and reagents

1. Papain dissociation system, which includes papain, DNase I, ovomucoid inhibitor, and EBSS (Worthington Biochemical Corporation, catalog number: LK003150)

2. Phosphate buffered saline (PBS) 10× concentrate (Sigma-Aldrich, catalog number: P5493)

3. PNU-282987 hydrate (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) (Sigma-Aldrich, catalog number: P6499)

4. BrdU (5-Bromo-2’-deoxyuridine) (Sigma-Aldrich, catalog number: 19-160)

5. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: A5670701)

6. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)

7. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148)

8. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881)

9. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

10. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418)

11. BD Horizon^TM brilliant stain buffer (BD Biosciences, catalog number: 563794)

12. Fc receptor binding inhibitor polyclonal antibody, eBioscience^TM (Thermo Fisher Scientific, catalog number: 14-9161-73)

13. Fluorescent antibodies and unconjugated blocking antibodies (see Table 1)

14. UltraComp eBeads^TM compensation beads (Invitrogen, catalog number: 01-2222-42)

    
    

    Table 1. Antibody panel used in this protocol

    Antibody	Fluorophore	Clone	Host	Company	Catalog number	Dilution (per 1 × 106 cells)
    Rhodopsin	Unconjugated	RET-P1	Mouse	Invitrogen	MAS-11741	3 μL
    Zenon kit	Alexa-405	-	-	Invitrogen	Z25013	-
    Thy 1.2	APC-eFluor-780	53-2.1	Rat	eBioscience	47-0902-82	1 μL
    Vimentin	Biotinylated	280618	Rat	R&D Systems	BAM2105	2 μL
    Streptavidin	BUV-395	-	-	BD Biosciences	BD 564176	2 μL
    Ki67	BUV-737	20Raj1	Mouse	eBioscience	367-5699-42	5 μL
    BrdU	PerCP-eFluor-710	BU20A	Mouse	eBioscience	46-5071-42	1 μL

Solutions

1. DMSO/PNU-282987/BrdU eyedrop solution (see Recipes)

2. DMSO/BrdU eyedrop solution (see Recipes)

3. 4% PFA (see Recipes)

4. FACS buffer with 4% FBS (see Recipes)

5. 0.4% Triton X-100 (see Recipes)

6. 0.1 M HCl (see Recipes)

1. DMSO/PNU-282987/BrdU eyedrop solution

   Caution: Appropriate PPE must be worn when handling chemicals.

   Reconstitute 50 mg of PNU-282987 in 1.66 mL of DMSO to make a 100 mM stock that can be stored at 4 for several months. Dissolve 5 mg of BrdU in 5 mL of DMSO to make a stock solution of 100 mg/mL BrdU that can be stored at -20 for several months. To make PNU-282987/BrdU eye drop solution, mix 9.8 mL of 1× PBS, 100 μL of 100 mM PNU-282987, and 100 μL of BrdU to achieve a final solution of 1 mg/mL BrdU and 1 mM PNU-282987. Store at 4 for one month.




2. DMSO/BrdU eyedrop solution

   Mix 9.8 mL of 1× PBS, 100 μL of DMSO, and 100 μL of BrdU to achieve a final solution of 1 mg/mL BrdU. Store at 4 for one month.




3. 4% PFA

   Caution: Preparation of PFA is hazardous. Avoid skin contact and inhalation. Use appropriate PPE and a chemical fume hood.

   Mix 4 g of PFA and 100 mL of 1× PBS in a media storage bottle containing a stir bar. Add drops of 2 M NaOH while the solution is stirring until a pH of approximately 7.4 has been reached or the PFA has completely dissolved. Please note the solution may become more acidic as PFA dissolves and the addition of more NaOH may be necessary. Place the bottle on a hot plate heated to 85 for approximately 20 min or until there are no visible particles. Allow the solution to cool at 4 . Add 1 M HCl drops until the solution reaches a pH of 6.9. Store at 4 for one week.




4. FACS buffer with 4% FBS

   Add 4 mL of FBS to 96 mL of 1× PBS. Store at 4 for one month.




5. 0.4% Triton X-100

   Add 4 mL of Triton X-100 to 96 mL of 1× PBS. Store at 4 for several months.




6. 0.1 M HCl

   Add 1.5 mL of 1 M HCl to 13.5 mL of MilliQ water for a final concentration of 0.1 M HCl. Store at room temperature for several months.

Laboratory supplies

1. 10 mm Petri dish (VWR, catalog number: 10799-192)

2. 90 mm Petri dish (Sigma-Aldrich, catalog number: P10903)

3. General lab supplies (pipettes, tips, tubes, etc.)

4. 5 mL Falcon round-bottom Polystyrene test tubes with 70 μm cell strainer (Fisher, catalog number: 08-771-23)

Equipment

1. Dumont #7 curved forceps (Fine Science Tools, catalog number: 11274-20)

2. Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10)

3. Spring scissors (Fine Science Tools, catalog number: 15000-00)

4. Scalpel blades #11 (Fine Science Tools, catalog number: 10011-00)

5. Scalpel handle (Fine Science Tools, catalog number: 10003-12)

6. Corning^® LSE^TM digital water bath, 6 L, 120 V (Corning, catalog number: 6783)

7. Fisherbrand^TM analog hotplate stirrer (Fisher, catalog number: FB30786160)

8. pH indicator strips (Sigma-Aldrich, catalog number: 1095350001)

9. VWR^® water jacketed CO incubator (VWR, catalog number: 10810-744)

10. Pipet-Aid (Corning, catalog number: 07-202-350)

11. Centrifuge (Thermo Fisher Scientific, model: Heraeus Multifuge X3)

12. Eppendorf benchtop centrifuge (for microcentrifuge tubes at room temperature)

13. Benchtop vortex

14. Bright-Line counting chamber, which includes slides (VWR, catalog number: 100503-092)

    Note: Alternatively, an automated cell counter can be used for this protocol.

15. BD LSRFortessa^TM cell analyzer (BD Biosciences)

Software and datasets

1. FlowJo^TM software v10.10 (BD Life Sciences) was used for flow cytometry data analysis

Procedure