Rapid Culturing of Primary Murine Melanocytes and Fibroblasts: Isolating Melanocytes and Fibroblasts from Whole Murine Skin Sample
Published: April 30, 2023
Abstract
Source: Murphy, B. M. et al. Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures. J. Vis. Exp. (2019)
This video describes the rapid method to isolate and culture the melanocytes and fibroblast simultaneously from the postnatal mouse pup’s skin. These cells can be maintained and manipulated in vitro to study skin cell biology, wound healing, and melanoma.
Protocol
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…
Offenlegungen
The authors have nothing to disclose.
Materials
| Ethanol, 200 proof | Fisher Scientific | 22032601 | |
| Antibiotic Antimycotic Solution (100x) | Sigma-Aldrich | A5955 | |
| McIlwain Tissue Chopper | Ted Pella | 10180 | |
| Tissue Chopper Blade | Ted Pella | 121-6 | |
| Tissue Chopper Plastic Disk | Ted Pella | 10180-01 | |
| Collagenase Type I | Worthington Biochemicals | LS004156 | |
| Porcine Trypsin | Sigma-Aldrich | 85450C | |
| Trypsin | VWR | VWRL0154-0100 | |
| G418 Disulfide | P212121 | LGB-418-1 | |
| Dulbecco's Modified Eagle Medium | Gibco | 12800-082 | |
| RPMI 1640 media | Sigma-Aldrich | R8758 | |
| Dulbecco's Phosphate Buffered Saline | Sigma-Aldrich | D8537 | |
| 6-well cell culture dish | Sigma-Aldrich | SIAL0516 | |
| 5 mL polystyrene round-bottom tubes | Fisher Scientific | 352008 | |
| 10 cm cell culture dish | Corning | 430167 | |
| Bovine Serum Albumin | Fisher Scientific | BP9706-100 | |
| Fetal Bovine Serum | Sigma-Aldrich | 12306C | |
| Corning Penicillin/Streptomycin Solution | Fisher Scientific | 30-002-CL | |
| Nutrient Mix F-12 Ham's Media | Sigma-Aldrich | N6760 | |
| Collagen from rat tail | Sigma-Aldrich | C7661 | |
| 0.2 µm PES sterile syringe filter | VWR | 28145-501 | |
| 40 µm cell strainer | Fisher Scientific | 22363547 | |
| 70 µm cell strainer | Fisher Scientific | 22363548 | |
| Cholera Toxin | Sigma-Aldrich | C8052 | |
| Horse Serum | Fisher Scientific | 26050088 | |
| HyClone L-Glutamine | Fisher Scientific | SH3003402 |
Diesen Artikel zitieren
Rapid Culturing of Primary Murine Melanocytes and Fibroblasts: Isolating Melanocytes and Fibroblasts from Whole Murine Skin Sample. J. Vis. Exp. (Pending Publication), e20357, doi: (2023).