Obtain approval from your institutional animal ethics committee before commencing this or any other study involving animals. Experiments performed in this protocol were approved by the Ohio State University’s Institutional Animal Care and Use Committee (IACUC, protocol #2012A00000134).

1. Protocol Preparation

NOTE: The following reagent preparation instructions are appropriate for the generation of 9 cm² melanocyte and fibroblast cultures from a single mouse. Refer to the reagent preparation guide in Table 1 for larger scale isolations.

1. Prepare 1.5 mL of Collagen Solution containing 50 µg/mL collagen in 0.1 M glacial acetic acid.
2. In a laminar flow cabinet, coat one well of a 6-well cell culture dish with 8 µg/cm² (1.44 mL) Collagen Solution. Ensure that the well is completely covered by the Collagen Solution. Incubate the dish at 37 °C for 3 h or at 4 °C overnight. Prior to use, wash the collagen-coated well twice with 1.35 mL of sterile phosphate buffered saline (PBS) (150 µL of PBS per 1 cm²).
3. Assemble the tissue chopper in a laminar flow cabinet by carefully placing a chopping disk on the tissue chopper platform and securing a sterile blade to the movable arm.
4. Prepare 3 mL of 1x Antibiotic/Antimycotic Solution by diluting 100x antibiotic/antimycotic stock solution in sterile PBS.
5. Prepare 3 mL of fresh Skin Digestion Buffer containing 10% fetal bovine serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 10 mg/mL collagenase type I, 0.25% porcine trypsin and 0.02 mg/mL deoxyribonuclease I in RPMI 1640.
6. Prepare 6 mL of fresh Melanocyte Media containing 10% fetal bovine serum, 7% horse serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 0.5 mM di-butyryl cyclic AMP (dbcAMP), 20 nM tetradecanoylphorbol 13-acetate (TPA) and 200 pM cholera toxin (CT) in Nutrient Mix F-12 Ham's media.
   NOTE: Concentrated dbcAMP, TPA and CT stock solutions can be made, aliquoted and stored for >1 year at -80 °C. Base media lacking these components can be stored for up to 1 month at 4 °C.
7. Prepare 4 mL of Fibroblast Media containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine in Dulbecco’s Modified Eagle Medium. This media can be stored at 4 °C for up to 1 month.
8. Prepare 40 mL of 4% Paraformaldehyde Solution by diluting 16% paraformaldehyde in PBS. Store any excess at 4 °C for 1 month or -20 °C for up to 1 year.
9. Prepare a 1% saponin stock solution by mixing 0.5 g of saponin in 50 mL of PBS at 37 °C until the saponin has completely dissolved. Sterilize the solution for long-term storage using a 50 mL syringe fitted with a 0.2 µm PES filter. The resulting saponin stock solution can be kept at 4 °C for 1 month.
10. Prepare 200 µL of 0.1% Saponin Solution per mouse by diluting 1% saponin stock solution in PBS containing 3% bovine serum albumin (BSA).
11. Prepare 1 mL of Viability Solution by diluting 1 µL of fixable viability dye in 1 mL of PBS.

2. Melanocyte and Fibroblast Isolation

1. Euthanize 0 to 4 day-old male and/or female C57Bl/6J pups by decapitation and remove extremities from the euthanized mice using surgical scissors.
2. In a laminar flow cabinet, briefly roll the trunk of each mouse in a sterile Petri dish containing 70% ethanol. Remove the trunk from the ethanol and place it into an empty, sterile Petri dish.
3. Using surgical scissors, sterilized in 70% ethanol, make an incision in the skin on the ventral side of the trunk starting from the neck to the tail. Peel the skin from the trunk of the mouse using sterile forceps.
4. Place the skin dermis side down in a 6-well dish containing 3 mL of 1x Antibiotic/ Antimycotic Solution and incubate at room temperature for 2-3 min.
5. Turn on the tissue chopper with the following settings in place: Slice thickness: 1 µm; Blade force: ~60% of the maximum; Speed: ~50% of the maximum.
6. Transfer the skin, dermis side down, to a sterile tissue chopper disk and pass the skin completely through the activated tissue chopper 3 times.
7. Transfer the homogenized skin to a sterile, 15 mL conical tube containing 3 mL of Skin Digestion Buffer. Mix the resulting suspension by pipetting up and down 10-15 times with a P1000 micropipette.
8. Cap the conical tube and incubate the sample in a 37 °C water bath for 15 min, inverting the tube every 3-5 min.
9. Pellet the cells in the skin homogenate by centrifuging the conical tube in a swinging bucket rotor at 750 x g for 5 min at room temperature.
10. Using a P1000 micropipette, slowly and completely remove the Skin Digestion Buffer being careful not to disturb the pellet.
    NOTE: A portion of whole skin can be saved at this stage and used as a control for Step 3: Confirmation of Cellular Purity. Strain these cells through a 70 μm cell strainer and then begin at step 3.5 for further processing.
11. Thoroughly resuspend the cell pellet in 1 mL of Melanocyte Media by pipetting up and down 15-20 times with a P1000 micropipette. Add the resulting cell solution dropwise to an uncoated well of a 6-well dish containing 1 mL of Melanocyte Media.
12. Place the plated skin homogenate in a tissue culture incubator set at 37 °C and 5% CO_2. Allow the cultures to incubate for 40 min.
    NOTE: During this time, some fibroblasts in the skin homogenate will adhere to the uncoated dish while the melanocytes and keratinocytes remain in suspension.
13. Transfer the culture supernatant from the uncoated dish to one well of a pre-washed, collagen-coated 6-well dish. Add G418 to the media such that the final concentration is 100 ng/mL.
14. Add 2 mL of Fibroblast Media to one well of the uncoated dish, now containing adherent fibroblasts.
15. Incubate both cultures overnight in a tissue culture incubator set at 37 °C and 5% CO₂.
16. Separately aspirate the media and any debris from each culture, 16-24 h after plating. Wash each dish twice with 1 mL of sterile PBS, and then add 2 mL of fresh Melanocyte Media plus 100 ng/mL G418 to the melanocyte culture and 2 mL of fresh Fibroblast Media to the fibroblast culture.
17. After the melanocyte cultures have been treated with G418 for 48 h, wash the cells twice with 1 mL of sterile PBS and add 2 mL of fresh Melanocyte Media without G418 to the culture.
    NOTE: As fibroblasts in the melanocyte culture continue to die off post-G418 treatment, wash the dish with sterile PBS to remove dead cells and add fresh Melanocyte Media. Melanocyte and fibroblast cultures should be passaged when they reach 70-80% confluency.

3. Confirmation of Cellular Purity

1. Aspirate the media from each culture and carefully wash each dish with 1 mL of sterile PBS.
2. Add 0.7 mL of 0.25% trypsin to each culture and incubate the cultures in trypsin at 37 °C and 5% CO₂ for 1 min.
3. Dislodge the cells by pipetting the trypsin against the bottom of the dish using a P1000 micropipette.
4. Add 0.7 mL of the appropriate media to each trypsinized culture and transfer the cell solution into a 1.5 mL microcentrifuge tube.
5. Pellet the cells by centrifugation at 750 x g for 2 min at room temperature. Carefully remove and discard the supernatant using a P1000 micropipette.
   NOTE: Steps 3.1-3.5 can be used to passage melanocyte and fibroblast cultures. The resulting pellet should be resuspended in the appropriate media and placed in a new, uncoated (fibroblasts) or pre-washed, collagen-coated (melanocytes) dish.
6. Resuspend the cell pellet in 0.5 mL of ice-cold PBS.
7. Enumerate and transfer 0.5 million cells to a pre-chilled 1.5 mL microcentrifuge tube.
8. Repeat step 3.5 and then resuspend the cells in 100 µL of Viability Solution. Incubate the cell suspension for 30 min at 4 °C in the dark.
9. Repeat steps 3.5-3.6 twice.
10. Repeat step 3.5, then fix the cells by resuspending the pellet in 100 µL of ice cold 4% Paraformaldehyde Solution. Incubate the suspension for 15 min at room temperature in the dark.
11. Repeat step 3.5 twice, each time resuspending the pellet in 0.5 mL of 3% BSA in 1x PBS.
12. Repeat step 3.5, then resuspend the cell pellet in 100 µL of 0.1% Saponin Solution. Incubate the suspension for 15 min at room temperature in the dark.
13. Repeat step 3.5, then resuspend the cell pellet in 100 µL of 0.1% Saponin Solution containing 0.5 µg of rabbit anti-gp100 antibody, 0.5 µg of rabbit anti-Fibroblast-specific protein 1 (FSP1) antibody and 0.025 µg of mouse anti-Cytokeratin 14 (K14) antibody. Incubate the suspension for 1 h at room temperature in the dark.
    NOTE: While the K14 antibody was purchased pre-conjugated to Alexa Fluor 647, the gp100 and FSP1 antibodies were conjugated to CF 555 and CF 488, respectively. The staining of control populations should also commence at this step. Control cell populations should be isolated from culture and processed as described in steps 3.5-3.12.
14. Repeat step 3.11 twice, to remove any unbound antibody.
15. Repeat step 3.5, then resuspend the cell pellet in 200-400 µL of 3% BSA in 1x PBS.
16. Pass the cell solution through a 40 µm cell strainer into a 5 mL polystyrene round-bottom tube and analyze the strained cells by flow cytometry.