Stable Transfection of Cells in Suspension

David L. Haviland, PhD dhavilan at imm2.imm.uth.tmc.edu
Sat Feb 26 09:37:02 EST 2000


On Sat, 26 Feb 2000, Frank O. Fackelmayer wrote:

Hi:

> Try electroporation, it works well for transfecting difficult cell lines.

Agreed.  Also, there are increasing reports of success with Fugene-6 with
boyant cells such as Jurkat.

> As to the separation of dead cells from live ones, this is hardly possible with suspension cells. One way to improve the ratio of live vs. dead cells is
> split them quite early after transfection to have only very small number of cells in each well (microtiter plate). The live ones will propagate, and will
> form clones.

Ah... maybe... if the culture  grows to a sufficient level, i.e, a T75's
worth.  They can be centrifuged, resuspended in a smaller volume and *if*
of lymphoid origin, they can be laid over a Ficoll gradient (~1.077).
I've done this with Jurkat, U937, and HL-60 transfectants with success.  

"Your milage may vary.." if attempted with boyant, non-lympohid cells -
one would have to try a small sample as a "look-see"...

Hope this helps,
David

==========================
David L. Haviland, PhD
Assistant Professor, Immunology
University of Texas, HSC - Houston
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX  77030
http://www.uth.tmc.edu/~dhavilan
713.500.2413-Voice
713.500.2424-FAX
==========================



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