In article <7vpm1a$bb9$1 at pegasus.csx.cam.ac.uk>,
C Coward <cc122 at mole.bio.cam.ac.uk> wrote:
> Greetings,
>> I am investigating the ability of a novel compound to act as a
> transfection aid for a macrophage cell line (J774A). Previously I have
> observed that the pSV-b-gal plasmid (Promega) poorly expresses b-gal
in
> this cell line (or poorly transfects it). I am looking for a reporter
> plasmid suitable for use in J774, preferably with a straightforward
assay
> method for the degree of transfection. Does anyone have any
suggestions?
In my hands (and others' in our lab) J774 has proven to be
nearly impossible to transfect. I have tried various lipids and
electroporation without success. I haven't found any papers
describing efficient transfection of J774 cells.
I would love to hear if you manage to have reasonable transfection
efficiency (even 5%) with some trick. I would use CMV-driven Green
Fluorescent Protein (GFP) reporter as marker for transfection.
With this you can easily estimate the relative amount of transfected
cells by immunofluorescence or flow cytometry (with 488 nm argon-laser).
Good luck!
Tapani Ronni, Ph.D.
UCLA School of Medicine/Howard Hughes Medical Institute
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