Reporter for macrophage (J774) transfection
Bernard Murray, PhD
spam at 127.0.0.1
Tue Nov 9 14:43:29 EST 1999
In article <808orm$i0h$1 at nnrp1.deja.com>, tronni at my-deja.com wrote:
> 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).
Ah, but if you have a FACS you could sort the transfected cells
and collect the green ones (100% transfection efficiency!).
For lower tech methods (not with this cell line) I have
co-transfected with an expression vector for a cell surface
protein and then sorted the transfectants by MACS using a
suitable antibody. Invitrogen sell/sold (?) a kit based upon
this - actually you transfect with an expression vector for a
single chain antibody and then sort with beads coated with
If J774 is appreciably phagocytic this may not work too well
(maybe try chilling the cells?).
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
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