Isolation and propagation of mouse embryonic fibroblasts and preparation of mouse embryonic feeder layer cells
| Autor: | Anna E. Michalska |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
KOSR
Pluripotent Stem Cells Mitomycin Cell Cell Culture Techniques Mitosis Cell Count Cell Separation Biology Cryopreservation Dna staining Mice Mycoplasma Pregnancy medicine Animals Embryonic Stem Cells Cell Biology General Medicine Fibroblasts Isolation (microbiology) Embryonic stem cell Coculture Techniques Cell biology medicine.anatomical_structure Cell culture Gamma Rays embryonic structures Immunology Female Feeder Layer Cells Developmental Biology |
| Zdroj: | Current Protocols in Stem Cell Biology |
| ISSN: | 1938-8969 |
| Popis: | To realize their potentials, embryonic stem (ES) cells must be maintained in optimal culture conditions that preserve their pluripotency and self-renewal capacity. Mouse embryonic fibroblasts (MEFs) are used to prepare a feeder cell layer that supports the growth of ES cells and the quality of feeders is crucial for the maintenance of undifferentiated ES cells in prolonged culture. The protocols provided in this unit describe aspects of isolation and expansion of MEFs and maintenance of established feeder cells. Preparation of mitotically inactivated feeder cell layer (treatment with mitomycin C or gamma-irradiation) is also described. In addition, a method for counting cell numbers and a simple method for detection of mycoplasma contamination by in situ DNA staining are also provided. Methodology described has been tested in a real laboratory environment and provides detailed information regarding resource and time requirements as well as critical parameters and troubleshooting. |
| Databáze: | OpenAIRE |
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