All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Melanocyte and Fibroblast Isolation
- 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.
- 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.
- 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.
- 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.
- 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.
- Transfer the skin, dermis side down, to a sterile tissue chopper disk and pass the skin completely through the activated tissue chopper 3 times.
- Transfer the homogenized skin to a sterile, 15 mL conical tube containing 3 mL of Skin Digestion Buffer (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). Mix the resulting suspension by pipetting up and down 10-15 times with a P1000 micropipette.
- Cap the conical tube and incubate the sample in a 37 °C water bath for 15 min, inverting the tube every 3-5 min.
- 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.
- 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 confirmation of cellular purity. Strain these cells through a 70 μm cell strainer and further process.
- 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.
- Place the plated skin homogenate in a tissue culture incubator set at 37 °C and 5% CO2. 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.
- 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.
- Add 2 mL of Fibroblast Media to one well of the uncoated dish, now containing adherent fibroblasts.
- Incubate both cultures overnight in a tissue culture incubator set at 37 °C and 5% CO2.
- 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. (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.
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)
- 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.