Isolation of tdTomato Expressing Inter-follicular Epidermal Melanocytes or Keratinocytes from Mouse Tail Skin

Materials and Reagents

1. Disposable gloves

2. Nair^® Wax Ready Strips for face (available at most drug stores or online)

3. 1 × Disposable scalpel (Fisher, catalog number: 02-688-79)

4. 1 × Ultra sharp general purpose razor blade (Fisher, catalog number: 13-812-236)

5. P20, P200, and P1000 Micropipette tips, sterile

6. 10 × 5 mL serological pipette (VWR, catalog number: 612-4945)

7. 4 × 10 mL serological pipette (VWR, catalog number: 612-4946)

8. 2 × 25 mL serological pipette (VWR, catalog number: 612-4947)

9. 4 × 15 mL conical tube (Corning, catalog number: 430790)

10. 3 × 50 mL Falcon^® tube (Corning, catalog number: 352070)

11. 3 × 30 mL Petri dish (Sarstedt, catalog number: 82.1194.500)

12. 2 × 10 mL syringe (Fisher Scientific, catalog number: 14-817-54)

13. 1 × DMSO-Safe Acrodisc Syringe Filter, 0.2 µm pore size, sterile (Pall, catalog number: 4433)

14. 2 × 16 G needle (BD Precision glide Needle, catalog number: 305197)

15. 1 × Falcon^® 40 µm Cell Strainer (Corning, catalog number: 352340)

16. 1 × 1.5 mL Eppendorf tube, sterile

17. 1 × Falcon^® 5 mL Round Bottom Polystyrene Test Tube, without Cap, sterile (Corning, catalog number: 352052)

18. Dispase II protease (Sigma-Aldrich, catalog number: D4693-1G)

19. Trypsin (1:250), powder (Thermo Fisher, catalog number: 27250018)

20. EDTA (0.5 M), pH 8.0, RNase-free (Thermo Fisher, catalog number: AM9260G)

21. Minimum Essential Medium (MEM) Eagle with HBSS w/o L-Gln, ("MEM") (Lonza, catalog number: BE12-127F)

22. Fetal Bovine Serum ("FBS"), suitable for cell culture, sterile-filtered (Sigma-Aldrich, catalog number: F1051)

23. eBioscience^® Fixable Viability Dye eFluor^® 450 (Thermo Fisher, catalog number: 65-0863-14)

24. RiboLock RNAse Inhibitor (Thermo Fisher EO0381) (For downstream RNAseq only)

25. Dispase solution (see Recipes)

26. Trypsin solution (see Recipes)

27. MEM + EDTA + 10% FBS Solution (see Recipes)

Equipment

1. 1 × Heat pad (Sunbeam brand, standard style, 12 × 0.5 × 15 inches, 3 temperature settings)

2. 1 × 37°C (5% CO₂) tissue culture incubator (Nuaire, catalog number: NU-S810)

3. 1 × Ice bucket and ice (Fisher, catalog number: S66360)

4. 1 × Analytical balance (Mettler Toledo, catalog number: AL54)

5. 1 × 1,000 µL micropipette (Gilson, catalog number: F123602)

6. 1 × 200 µL micropipette (Gilson, catalog number: F123601)

7. 1 × 20 µL micropipette (Gilson, catalog number: F123600)

8. 2 × Dumont #55 fine straight forceps (Fine Science Tools, catalog number: 11255-20)

9. 1 × Stereomicroscope (Leica, catalog number: MS5)

10. 1 × Dumont #7 fine curved forceps (Fine Science Tools, catalog number: 11274-20)

11. 1 × Swing bucket centrifuge with temperature control from 4 to 18°C (Eppendorf, model: 5804R)

12. Mouse anesthesia and euthanasia equipment, as specified by the investigator's Institute's Animal Care Ethics board and with Institute approval

13. Biosafety cabinet, Class II Type B2, if cells are to be cultured following isolation (Nuaire)

14. A cell sorter with the required lasers and detectors: 460/50 (405) and 585/29 (561) (e.g., Cytoflex LX Analyser, Beckman Coulter)

Procedure

Mouse Anesthesia Protocol

1. Obtain Institute approval before working with mice.

2. Use a reliable gas scavenging system to collect, remove, and dispose of waste anesthetic gases (fume hood, vacuum waste gas line, etc.) for the researcher's safety.

3. Place mouse into an inhalation chamber. Introduce 5% isoflurane in 1.0 L/min oxygen into the chamber, and observe the animal until it has lost its righting reflex.

4. Remove the mouse from the inhalation chamber, and attach a nose cone to continue to provide 5% isoflurane in 1.0 L/min oxygen. Place mouse on top of insulating material, such as a layer of paper towels, so that the mouse does not lose body heat as quickly, which happens faster if the work surface is made of metal.

5. Apply a corneal lubricant on a q-tip to the mouse’s eyes, to protect them.

6. Once the breathing rate of the mouse is less than 60 breaths per minute, check for a toe pinch response. When there is no response, carry out the waxing procedure.

7. When finished waxing, remove the nose cone. Place the mouse into a prepared recovery cage. This is a clean cage lined with a clean absorbable substrate (e.g., paper towel). The recovery cage should be placed on the heat pad set to low.

   Note: Rodents who are recovering from anesthesia should not be placed directly onto normal cage bedding material. Placing unconscious mice onto bedding poses several risks, including asphyxiation due to inhalation of bedding pieces, and damage to the corneas until the return of the blink response.

8. Monitor the mouse as it recovers from anesthesia, in a quiet area. Once it is fully recovered, the mouse can be returned to its home cage.

   
   

1. Waxing one day before cell collection

   1. Turn the heatpad on at the lowest setting to warm up.

   2. Cut four small rectangular pieces of Nair^® wax strips (40 mm × 5 mm), leaving a wax-free border on one narrow end of each strip, in order to hold on to the strip without touching the wax.

   3. Place mice under anesthesia for tail hair removal. We use a nose cone isoflurane inhalant system. For reference, our protocol for recovery anesthesia is included above. However, the exact method of anesthesia is not likely to affect the success of the experiment, and the procedure should be carried out according to the Animal Care Ethics board regulations that govern the institute where the research is being conducted.

   4. Warm two wax strips slightly by placing the strips on the heat pad for 20 s. This should warm the strips to body temperature (37°C).

   5. Gently remove clear plastic from the strips and place one strip on the top of the tail and one on the underside of the tail. The wax free area of the strip should be oriented so that it is placed towards the tip of the tail.

   6. Smooth each strip gently downwards with your fingers to make sure that it is properly adhered.

   7. Wait 20–25 s and then remove the wax strips with a quick gesture, in the direction that is towards the head of the mouse.

   8. Repeat steps A4–A7, but this time place the remaining two wax strips on the lateral sides of the tail.

      Note: The skin should be light pink, clean and without any injury. If some traces of wax remain on the skin, mouse self-grooming will remove it overnight.

      
      

2. Cell collection procedure

   1. Sterilize forceps before use.

   2. Make the dispase solution following the instructions located in the Recipes section.

   3. Euthanize the mouse according to the Animal Care ethics regulations that govern the institute where the research is being conducted. At the University of BC, this is isoflurane anesthesia, followed by CO₂, then cervical dislocation. Using an alternative method of euthanasia is not likely to affect the success of the experiment. Process the skin as soon as possible after euthanasia.

   4. Place the mouse on its back and, holding the tail base with forceps, use a scalpel to cut along the ventral midline from the tail base to the tail tip (Figure 2A) (Video 1).
      
      

      
      Video 1. Tail skin incision technique.

      
      

      This video was made at the University of British Columbia, according to guidelines from the University of British Columbia on Animal Care, and approved by the Animal Research Ethics Board of the University of British Columbia, under protocol #A19-0148.

      
      

      
      Figure 2. Isolating mouse tail skin from bone. (A) Forceps and a scalpel are used to cut the mouse tail skin along the ventral midline. (B–C) A razor blade is used to remove the tail and cut it into smaller segments about 0.5 cm in length. Ruler shown in mm. (D) Tail skin is peeled off of the bone and briefly set on glove, to reduce curling.

      
      

   5. Cut tail off and divide it into four segments (about 0.5 cm × 0.5 cm each) with a razor blade (Figure 2B–C). Discard the tip of the tail, where it becomes narrow.

   6. Using the skin incision made in step B3, peel the skin from the tail bones.

      After being removed from the bone, tail skin tends to roll in on itself. In order to flatten it, it is recommended that the skin be placed flat for about 30 s (e.g., on the back of a gloved hand) (Figure 2D).

   7. Place flattened tail skins in the "dispase" Petri dish. Ensure the epidermis side of the skin is facing up, and the skin is floating and not submerged (Figure 3).

      
      

      
      Figure 3. Tail skin floating in dispase solution. (A) Birds eye and (B) horizontal view of tail skin floating in dispase solution.

      
      

   8. Incubate the Petri dish with the tail skins in the dispase solution in a tissue culture incubator at 37°C (5% CO₂) for 35 min.

   9. Meanwhile:

      1. Remove Fixable Viability Dye from -80°C and thaw it on ice.

      2. Add 2 mL of MEM to a Petri dish and label it “for trypsin”.

      3. Make the trypsin solution using the instructions located in the Recipes section.

      4. Make the MEM + EDTA + 10% FBS solution using the instructions located in the Recipes section, and keep on ice until use.

      5. Transfer 2 mL of the MEM + EDTA + 10% FBS solution to a 50-mL Falcon tube labeled “cells 1”, and place the tube on ice.

      6. Chill an empty 15-mL conical tube labeled "cells 3" on ice.

   10. Remove the Petri dish with skin pieces in dispase solution from the incubator.

   11. Using a 5-mL serological pipette, remove the dispase solution and discard.

   12. Add, and then remove, 1 mL of MEM twice, to wash the skin pieces. They will float.

   13. Add 1 mL of MEM to the dish.

   14. Using a pair of fine forceps, split the epidermis from the dermis (Figure 4) as follows:

       1. Remove one piece of skin and place it in a new empty Petri dish. Use one pair of forceps to clamp onto the epidermis while the other forceps peels the white dermis layer away. Starting in a corner works best for peeling the entire segment in one motion.

       2. A stereomicroscope will aid in discerning epidermis from dermis.

          
          

       
       Figure 4. Splitting dermis from epidermis. (A) A pair of fine forceps is used to split the dermis from epidermis starting in one corner and (B) following through in a single motion. (C) The end result, with dermis in the left Petri dish and epidermis on the right.

       
       

   15. Immediately after the dermis has been removed, transfer the epidermal layer to the Petri dish labelled “for trypsin,” currently holding 2 mL of MEM. The epidermis should be floating, with the underside of the epidermis in contact with the solution. Discard the dermis.

   16. Repeat steps B14 and B15, until the dermis from all skin pieces has been removed and the epidermis placed in the "for trypsin" dish.

   17. Using two 5-mL serological pipettes, remove the MEM from the Petri dish holding the epidermis and replace it with 3 mL of the trypsin solution, making sure the pieces of epidermis are still floating.

   18. Incubate the Petri dish with the pieces of epidermis in the trypsin solution in a tissue culture incubator at 37°C (5% CO₂) for 5 min.

   19. Remove the Petri dish containing the epidermal pieces in trypsin solution from the incubator.

   20. Inhibit trypsin activity by adding 3 mL of cold MEM + EDTA + 10% FBS solution to the dish, with a 5-mL serological pipette. Swirl to mix.

   21. Then, use the same serological pipette to collect 4 mL of the solution from the dish and add it to the chilled conical tube labeled “cells 1” from step B9.

   22. Use the following steps to release and collect cells from the epidermis:

       Pick up one piece of epidermis using a fine forceps #55 and scratch the underside of the scales once or twice using a pair of curved forceps #7. This will release some cells into the 2 mL of solution in the Petri dish. Epidermal cells at this stage appear white. Use a stereomicroscope to better visualize the process (Video 2, before & after epidermal scratching).

       
       
       
       Video 2. Scale scratching technique
       
       

       1. Use the 1,000 μL micropipette to transfer 1 mL of the solution in the Petri dish, including the released cells, to the Falcon tube labeled “cells 1”.

       2. Immediately add 1 mL of fresh, cold MEM + EDTA + 10% FBS solution to the Petri dish.

       3. Repeat this process: Scratch and transfer cells from each piece of epidermis three times. The final volume transferred into "cells 1" will be 14 mL (which will be added to the 4 mL already in the tube). If less volume is used, cell viability will suffer.

   23. Using a 10-mL syringe and a 16 G needle, pipette the "cells 1" solution up and down 10 times, slowly, to limit the formation of air bubbles in the syringe. This process disrupts the cell clumps and separates the cells.

   24. Pass the cell solution through a 40 µm cell strainer into a new 50-mL Falcon tube labeled "cells 2", to remove any clumps of cells that did not dissociate in step B23.

   25. Using a 10-mL serological pipette, rinse the cell strainer mesh with 6 mL of cold MEM + EDTA + 10% FBS solution into the "cells 2" tube.

   26. Centrifuge the "cells 2" tube at 300 rcf (× g) and 18°C for 3 min. A pellet should form.

   27. Lower the temperature of the centrifuge to 4°C.

   28. Gently remove as much supernatant as possible from "cells 2" without disturbing the pellet, using a 25-mL serological pipette. Discard the supernatant.

   29. Add 1 mL of fresh cold MEM + EDTA + 10% FBS solution, and gently resuspend the cells.

   30. Transfer cells to the pre-chilled 15-mL conical tube labeled "cells 3" from step B9.

   31. Add 1 µL of Fixable Viability Dye to "cells 3" and gently mix.

   32. Incubate "cells 3" on ice for 15 min.

   33. Use a 10-mL serological pipette to add 7 mL of fresh, cold MEM + EDTA + 10% FBS to "cells 3".

   34. Centrifuge "cells 3" at 300 rcf (× g) and 4°C for 3 min.

   35. Using a 10-mL serological pipette, remove supernatant from "cells 3," leaving about 0.2 mL of liquid above the pellet.

   36. Gently resuspend the pellet in the remaining 0.2 mL of liquid, and then transfer cells to an Eppendorf tube. Keep the Eppendorf tube with cells on ice until FACS sorting, which should be done as soon as possible.

   37. Add 20 µL of fresh, cold MEM + EDTA + 10% FBS solution to a 15-mL round bottom polystyrene test tube, which will be used to collect the tdTomato-positive cells during FACS.

       1. If RNA sequencing is the final goal, also add 1 µL of RiboLock RNase Inhibitor.

   38. FACS sort for tdTomato positive cells at 581 nm, while excluding Fixable Viability Dye (pacific blue) positive dead cells, which are fluorescent at 405 nm.

Notes

1. To succeed in separating the dermis from the epidermis with the dispase solution, tail pieces should be no larger than 0.5 × 0.5 cm.

2. If the cells are intended for cell culture, then whenever possible, the procedure should be conducted inside a biological safety cabinet using sterile technique. In our experiments, antibiotics were not added to the media until after cells were isolated by FACS and plated for cell culture. Others may wish to try adding antibiotics earlier, if there are problems with contamination in the culture.

3. In the first experiment, tdTomato-negative epidermis should be used as a control to set the gates for sorting tdTomato-positive cells.

4. Please see Urtatiz et al. (2021) for further details on our melanocyte primary culture conditions, if that is the desired application. Other published cell culture protocols will also be suitable.

Data analysis

Cell sorting from K14-cre; tdTomato mice yielded the results in Figure 5. The epidermis contains many dead keratinocytes, which are pushed upwards and shed as part of the normal process of skin renewal. Hence, there is a large population of pacific blue-positive (dead) cells. The target population is shown in red. These are tdTomato-positive, pacific blue-negative cells (keratinocytes). The yield of these cells is about 400,000 per tail.

Figure 5. Example results of FACS performed on dissociated K14-cre; tdTomato mouse tail epidermis. This experiment shows the isolation of keratinocytes. A–C) Cells were first analyzed and gated using various forward scatter (FSC) and side scatter (SSC) properties, as is standard for FACS. The P1 and P2 gatings were used to remove debris. The P3 gating was used to remove cell doublets. D) The P3 population of cells (singlets) was then gated and analyzed for the pacific blue viability dye (y-axis) versus tdTomato (x-axis) fluorescence. The tdTomato-positive, pacific-blue negative cells (shown in red throughout Figure 5) are live keratinocytes, the target population. The pacific-blue positive cells (orange and purple) are dead cells, some of which are Tomato-positive (purple) and some of which are not (orange). Dead cells will eventually lose tdTomato fluorescence. The tomato-negative, pacific-blue negative cells (blue) are live cells that did not express tdTomato. These make up a small percentage. E) A heat map of the same cells shown in panel D. F) A FSC versus SSC heatmap of just the live, tdTomato-positive population, which was gated from the other cells in panels D/E.

Cell sorting from Mitf-cre; tdTomato mice yielded the results in Figure 6. The target population is shown gated as population P5 (green). These are tdTomato-positive, pacific blue-negative cells (melanocytes). There are far fewer melanocytes in the inter-follicular epidermis than there are keratinocytes. In this experiment, live tdTomato-positive melanocytes made up 0.64% of the cells. Previous studies have estimated that melanocytes account for ~1.5% of the total cells in the IFE (Glover et al., 2015). Typically, we process the epidermis from two mouse tails together to get a yield of ~25,000 melanocytes for downstream RNAseq or primary cell culture applications.

Figure 6. Example results of FACS performed on dissociated Mitf-cre; tdTomato mouse tail epidermis. This experiment shows the isolation of melanocytes. A–C) Cells were first analyzed and gated using various forward scatter (FSC) and side scatter (SSC) properties, as is standard for FACS. The P1 and P2 gatings were used to remove debris. The P3 gating was used to remove cell doublets. D) The P3 population of cells (singlets) was then gated and analyzed for the pacific blue viability dye (y-axis) versus tdTomato (x-axis) fluorescence. The tdTomato-positive pacific-blue negative population (P4) contains the target cells, but also some debris or doublets. The vast majority of cells are tdTomato-negative (purple). E) A heat map of the same cells shown in panel D. F) A FSC versus SSC heatmap of just the P4 population, which was gated in panels D/E. A tight grouping of cells forms the P5 population indicated in green throughout Figure 6. This is the target population of the experiment (live single melanocytes). The P6 population likely represents debris or doublets that were missed during the P1–P3 gating.

Recipes

1. Dispase solution

   1. Add 5 mL of MEM to a 15-mL conical tube using a 5-mL serological pipette.

   2. Weigh out 0.01 g of dispase and add it to the MEM (final concentration = 0.2%).

   3. Mix by manually shaking the tube until the dispase is dissolved.

   4. Transfer the dispase solution to a Petri dish.

   5. Label the dish "dispase".

2. Trypsin solution

   1. Add 9.982 mL of MEM to a 15-mL conical tube.

      Use a 10-mL serological pipette to add 9 mL of MEM and a 1,000-µL micropipette to add the remaining 982 µL.

   2. Weigh out 0.01 g of trypsin and add it to the tube (final concentration = 0.1%).

   3. Mix by manually shaking.

   4. Add 18 µL of 0.5 M EDTA to the tube.

   5. Mix by manually shaking.

   6. Filter the trypsin solution into a new 15-mL conical tube using a 10-mL syringe with a syringe filter.

   7. Label the tube "trypsin solution".

3. MEM + EDTA + 10% FBS Solution

   1. Add 26.944 mL of MEM to a 50-mL Falcon tube.

      Use a 25-mL serological pipette to add 25 mL and a 1,000-µL micropipette to add the remaining 1,944 µL (1 × 1,000 µL and 1 × 944 µL).

   2. Add 54 µL of 0.5 M EDTA to the MEM using a 200-µL micropipette.

   3. Use a 5-mL serological pipette to add 3 mL of FBS to the MEM + EDTA.

   4. Mix by manually shaking.

   5. Keep on ice until use.

Acknowledgments

We thank Dr. Greg Barsh for the gift of the Mitf-cre mice. This work was supported by the Canadian Institutes of Health Research (MOP-79511 to C.D. Van Raamsdonk).

The original research paper for this protocol was published in eLife, DOI: 10.7554/eLife.71825 (Urtatiz et al., 2021).

Competing interests

We have no competing interests to declare.

Ethics

This work was carried out under the approval of the UBC Animal Care Committee under A19-0148, approved on 7/30/2019, with annual renewals.

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