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
antigen.
If J774 is appreciably phagocytic this may not work too well
(maybe try chilling the cells?).
Bernard
--
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
