Fibroblast Blues

Jerry Woodward woodward at seqanal.mi.uky.edu
Wed Jul 21 09:08:54 EST 1993


Re-clone your hybridoma line.  Go back to the earliest frozen stock you have,
get it growing and then do limiting dilution cloning:  Set up 96 well plates
with 100, 10, and 1 cell per well.  Use irradiated mouse spleen cells as 
feeders---5 x 10e5 per well.  Feed every three to four days, and you may
want to add additional irradiated spleen cells.  You should see growth in 
positive wells after 12-20 days.  In the plates with 20-30% of the wells
showing hybridomas, these are likely to be derived from a single clone.
You can sometimes tell visually if the clone is not disturbed by feeding.
Finally, you need to test the supernatants from expanded clones for 
antibody production.
Also, you might want to shoot some of your cells into a mouse for ascites. 
You will probably be able to propagate the line this way if you loose the
in vitro line.
J. Woodward
Univ. of Kentucky



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