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Mouse Embryonic Stem Cell Maintenance for Differentiation

HI Hogune Im

Published: Oct 20, 2011 DOI: 10.21769/BioProtoc.145 Views: 17011

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Abstract

Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a protocol to maintain mouse stem cells that will be used for hematopoietic lineage differentiation assay in the lab.

Materials and Reagents

  1. Mouse embryonic stem cells (ES cells)
  2. STO cells
  3. Fetal calf serum (FCS) Gemini (pre-selected) (catalog number: 100-500 )
  4. ATLAS biologicals, Summit (Summit Biotechnology, catalog number: FP-200-05 )
  5. DMSO (Sigma-Aldrich)
  6. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14190-144 )
  7. Gelatin (STEMCELL Technologies, catalog number: 0 7903 )
  8. Monothiolglycerol (MTG): (Sigma-Aldrich, catalog number: M-1753 25 ml )
  9. IMDM media (Life Technologies, Gibco®, catalog number: 12440-053 )
  10. CCE cells (ATCC, catalog number: SCRC-1023 TM)
  11. Mitomycine C (Sigma-Aldrich, catalog number: M0503 )
  12. Penicillin/Streptomycin (Pen/Strep) (10,000 U) (Life Technologies, Gibco®, catalog number: 15140-122 )
  13. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
  14. Trypsin /EDTA (Lonza Group, catalog number: 17-161E )
  15. HEPES buffer (Life Technologies, Gibco®)
  16. MTG
  17. BME
  18. Eosin
  19. Iscove’s modified dulbecco medium (IMDM) (10% FCS) media
  20. β-Mercaptoethanol (BME) (see Recipes)
  21. ES-DMEM media (see Recipes)
  22. STO media (see Recipes)
  23. ES freezing media (see Recipes)
  24. Leukemia inhibitory factor (LIF) (screened) (see Recipes)
  25. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 12100-046 , 1 L/pack) (Liquid, catalog number: 11965 ) (see Recipes)

Equipment

  1. 1 L with volumetric flask
  2. 0.45 micron filter
  3. 0.22 micron filter
  4. T25 flask
  5. T75 or T175 flask
  6. Water bath

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Stem Cell > Embryonic stem cell > Maintenance and differentiation

Developmental Biology > Morphogenesis

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