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Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line

YP Yuqiong Pan

Published: Oct 5, 2011 DOI: 10.21769/BioProtoc.142 Views: 16968

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Abstract

Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyrc-2J (c2J). Coat-color chimera production was high using c2J blastocysts while FVB blastocysts produced a low number of chimeras [PubMed: 11730008].

Materials and Reagents

  1. Cells and cell line:
    1. C57BL/6J (B6) mouse blastocyst (PubMed: 11730008 )
    2. #693 C57BL/6 mouse ES cell line (ATCC, catalog number: SCRC-1002 ™)
    3. CF1 mouse embryonic fibroblast (MEF) feeder cells (self-made from day 12.5 CF1 strain mouse embryos) or other mouse feeder cells such as DR4 (Applied StemCell, catalog number: 1013 )

  2. Medium and growth factors:
    1. DMEM (Life Technologies, Invitrogen™, catalog number: 11995-073 )
    2. Fetal bovine serum (FBS), Qualified (US) (Life Technologies, Invitrogen™, catalog number: 26140-079 )
    3. 100x MEM non-essential amino acids (NEAA) (Life Technologies, Invitrogen™, catalog number: 11140-050 )
    4. 1,000x 2-Mercaptoethanol, liquid (Life Technologies, Invitrogen™, catalog number: 21985-023 )
    5. 100x L-Glutamine (20 mM), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
    6. Penicillin/streptomycin (Pen/Strep), liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
    7. 1,000 U/ml ESGRO® mouse leukemia inhibitory factor (LIF) (Merck KGaA, catalog number: ESG1107 )
    8. TrypLE™ express stable trypsin-like enzyme with phenol red (Life Technologies, Invitrogen™, catalog number: 12605-028 )
    9. Gelatin from porcine skin-BioReagent, Type A, powder (Sigma-Aldrich, catalog number: G1890-100G )
    10. 1x PBS (pH 7.4), liquid (Life Technologies, Invitrogen™, catalog number: 10010-049 )
    11. Ethanol
    12. 0.25% gelatin (see Recipes)
    13. Mouse ES cell medium (see Recipes)

Equipment

  1. Incubator: 5% CO2 in humidified air, 37 °C (Thermo Fisher Scientific)
  2. Centrifuges and rotor (Thermo IEC) 
  3. BD Primaria* Tissue Culture Dishes, 100 x 20 mm, 08-772-4F (Thermo Fisher Scientific, catalog number: 353803)
  4. 10 cm tissue culture dish
  5. Water bath
  6. 0.22 µm filter

Procedure

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

Category

Stem Cell > Embryonic stem cell > Maintenance and differentiation

Developmental Biology > Cell growth and fate

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