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RESEARCH DIVISION Laboratory Manual

 


 

Harvesting of Cells for Blastocyst Injection

Culture should be 2 days old and colonies should be of medium to large size. This is important as cells need to be in log growth phase.

Two small (25cm2) flasks are required, one is harvested in morning and the other approx. 2 hours later, if there are sufficient blastocysts.

  1. Remove medium from flask.
  2. Wash with 5 mls PBS.
  3. Wash with 5 mls PBS/EGTA, remove PBS _ 37C tray for approx. 3 min or until cell boundaries can be seen.
  4. Add 1 ml TRYPSIN/EDTA 0.25%, rock backwards and forwards for 1 min - by this time all the ES Cell colonies and feeders should be detached.
  5. Using a 1 ml pipette disperse cells until single cells can be seen - no longer than 1 min.
  6. Neutralise the TRYPSIN/EDTA with 1 ml medium _ 10 ml tube _ count in hemocytometer.
  7. Centrifuge cells 3 min, speed 3 (1000 rpm), resuspend cells at a concentration of 106/ml in DMEM with hepes.
  8. Add 0.05 ml cells to 1 ml DMEM with Hepes (see Media for embryo manipulation and culture) with 3000U DNA'ase = 100 cells/ul this is the correct concentration for injection. DNAse is in aliquots in -20C, add to 1ml of DMEM with Hepes and fliter before adding to cells.

NOTES:

  1. TRYPSIN/EDTA is normally 0.05% TRYPSIN to make up to 0.25%, 1.6 mls of 2.5% Trypsin is added to 20 mls 0.05% Trypsin/EGTA.
  2. DNase I (Deoxyribonuclease 1, E.C. 3.1.21.1, Boehringer Mannheim Cat. No. 1284 932) 100 mgs @ 2000U/mg = 2 x 105 U. Dissolve in 2 mls PBS = 100U/ul. Add 30ul (3000U) to 1ml M2 medium. Make 30ul aliquots of DNase I and store at -20C, otherwise it degrades into filamentous bits that block the injection needle.
  3. DMEM with Hepes -
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998