One of our users has sorted based on DNA content. They use 10ug/ml
Hoechst 342 and stain for one hour at 37C. With adherent cultures,
they have the cells in log phase (50-60% confluent). With suspension
cultures they stain at no more than 3million/ml, but the cells are
grown at 500,000 to 800,000/ml then concentrated for the staining.
After the cells are harvested they are resuspended in media with
10ug/ml H342 at 10 to 20 million/ml. The sample block is kept cold
during the sort and the cells are sorted into tubes that are also on
ice to block progression thru the cell cycle. Our user isolates the
DNA from the cells after the sort so I can't respond to the viablity
of the cells at that point. It is important that your UV power is low
(50-100mW) so you don't fry the cells. You should check that the time
of staining and cell concentration during the staining are optimal for
your cell line and you can check at that time if your cells are still
viable. Good Luck.
Lucy Brown
Beckman Research Institute/City of Hope
626-359-8111 X3306
lbrown@coh.org
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