HEp-2 Transfections

[Rob Brazas]

Hi Everybody,

I've been trying to transfect HEp-2 cells without much success. I need to
do some immunofluorescence using the transfected cells, and I want my
transfection efficiency to be on the order of about 10% or better. I've
tried electroporation, CaPO4, Liopofectamine and DOTAP. I've really only
been able to get about 5 -10 cells per plate (6 cm) transfected. I've been
using an SV40 early promoter driven Beta-gal reporter to assay for
transfection efficiency about 36 hr post transfection using X-gal. I must
admit that I've only tried minimal optimization of the lipofectamine and
DOTAP (different concentrations of reagent with about 5 ug of DNA and a set
6 hr incubation).

The bottom line is that just want a protocol that is known to work well on
these cells. Thanks for the help, I hope!

Rob



Robert M. Brazas
Department of Microbiology, HSE401
University of California, San Francisco
San Francisco, CA  94143-0414

Phone#  415-476-4627
Fax#      415-476-0939
email      brazas at cgl.ucsf.edu