293 Transfection

Iolaus at my-dejanews.com Iolaus at my-dejanews.com
Thu Jun 4 10:12:01 EST 1998


I am transfecting the 293 cells with Lipofectamine Plus.  In my analysis,
whether staining or harvesting for quantitation, it get a lot of cells
lifting.  I have found that warmed PBS does alleviate some of the cell loss,
but I was wondering if anyone has tried using a cocktail of matrix proteins,
or any other technique with better success

Kevin


In article <6l4q14$8gd at mserv1.dl.ac.uk>,
  wgschech at med.uni-tuebingen.de wrote:
>
> Hi.
>
> What transfection method do you use?
>
> I just can tell you that my 293s are sensitive, too. I'm using
> different calcium phosphate methods (BBS and HBS, with and without
> glycerol shock). Cells are cultivated on polystyrene plates without
> any previous coating.
> I avoid agitation when washig and try to treat them as gently as
> possible.
>
> Since I use 293s only for testing plasmids and to observe "crude
> effects" (since they are transformed it's unclear one is able to
> transfer results on "normal" (explanted) cells (renal mesangial cells
> in my case), I sometimes scrape the cells off, remove them
> with media, entifuge and analyze the pellet.
>
> You also might try using collagen (we use HOAc extract from rat
> tails, mostly type IV if I remember correctly and dry in vacuo) or
> gelatine (rinse dishes with .15% or so, in water, you even then
> could dry the dishes then in vacuo) for coating. Probably cheaper
> than this high end stuff and surely worth a try.
>
> That's what we did with our mesangial cells before we noted that they
> grow on our new brand of dishes fine without anything. Maybe
> changing this will help, too.
>
> Good luck!
>
> Wolfgang
>
> > I am transfectng 293 cells on poly-lysine coated plates, and am
> > still having cells detach.  i was woundering if anyone has any
> > "tricks" in preventing this from occuring.  I was considering on
> > making a cocktail of Fibronectin, Collagen, and Vitronectin to
> > include in plating media. Any suggestions would be welcomed.
> >
> > Thanks
> >
> >
> >
> -----
> usual disclaimers apply * This message is RNAse free - please don't touch!
> -----
> Wolfgang Schechinger
> University of Tuebingen, Germany
> email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
> -------
>


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