methylcellulose plating problem

Scott Brown via methods%40net.bio.net (by SBrown from ccia.unsw.edu.au)
Sun Feb 15 20:23:26 EST 2009


Hi,
 
This may be a long shot but i am banging my head against the wall over it. I have been doing some methocult plating, basically i take normal Nalm-6 cells, add 1.6 mls methocult, 1.2 mls FCS, 1.2mls IMDM media and then i add dexamethosone at a conc. of 8uM.
Last year this would kill the majority of the Nalm-6 cells (EC50 = 6nM), there were always some colonies that would grow through so i added some methotrexate to try and decrease the formation of colonies.
For the past 6 months I have been getting equal numbers of colonies in my control (the nalms) as well as my experimental population of cells. I've checked my conc. of dex, repeated MTTs to confirm the EC50, repeated the plating several times but continue to get too many colonies being formed in the control. Previous plating showed a distinct difference in the amount of colonies formed in the control and the experimental situation.
 
Does anyone have any ideas as to what may be going on or any suggestions for an experiment i may be able to do to get to the bottom of this?
 
cheers
 
Scott 
 
Scott Brown
PhD Candidate
Molecular Carcinogenesis Program
Children's Cancer Institute of Australia for Medical Research
High Street (PO Box 81)
RANDWICK NSW 2031
AUSTRALIA

Phone: +61 2 9382 1829
Fax: +61 2 9382 1850
Email: sbrown from ccia.unsw.edu.au
Web: www.ccia.org.au <http://www.ccia.org.au/>  
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Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering.

The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message.



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