separation of dead/alive T cell hybridoma cells

John Ladasky ladasky at leland.Stanford.EDU
Wed Sep 25 12:53:55 EST 1996

In article <52bh2u$ab7 at>,
Paula Lavery  <plaver at PO-Box.McGill.CA> wrote:
>>   bboverduin at (Bert Overduin) writes:
>>  I am looking for a method to separate alive and dead mouse suspension T
>>  cell hybridoma (3DO) cells other than fluorescence activated cell sorting.
>>  I am not very familiar with animal cell biological techniques, but guess
>>  there must be a way to accomplish this. Answers and or hints can dbe
>>  directly sent to me or posted on the newsgroup.
>>  Bert Overduin
>>  CEPRAP / UC Davis
>>  bboverduin at
>Try spinning your cell suspension over a Ficoll-Hypaque gradient.  I have
>done this to clean up both hybridoma cells as well as preparations of
>activated cells.  
>Cells harvested from the interface are usually 95-100% viable.
>You will probably lose some cells in the process, so if cell numbers are
>crucial you may
>want to try another method first.

	I have only been able to get this method to work with fairly fresh
peripheral blood mononuclear cells.  When I tried it with a cell line that
I had preincubated for a few days in serum-free medium (which induced some
cell death), no cells passed into the Ficoll layer.  All my cells, live,
dying, and dead, sat on top of the interface.  Viability in the recovered
fraction was identical to the initial cells (about 70%).

	Does anyone know why?

Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green

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