Transfection by Ca-phosphate of a B-cell line
kalsriv at mailcity.com
Mon Dec 10 16:49:38 EST 2001
Just like you, I was also new to the world of transfection, few months
ago. But after months of trial and error I have also started getting
some good transfection these days. So I think I can share my limited
knowledge on this.
Well actually, Maniatis and other literature reveal that cells exhibit
different levels of toxity to the extracellular calcium cocentration.
For example, I just leave the transfection cocktail for overnight when
I trasfect the 293T cells and change the media next day, but change
the media within four hours when I work with COS7 cells.
So you have to, sort of check, the tolerance of B cells for calcium
(which according to my knowledge is very less, because B Cells show
apoptosis with increased level of calcium). My suggestion is also that
you must try lipofectamine, PEI and most probably the
electro-transfection (I am not sure about it).
Consult also for pcDNA 3.1 as toxic thing for B cells. Check the pH
of HBS if it is not too alkaline or acidic (I make it 7.04). Last, it
may also depend upon the gene that is introduced.
ruben.varela-calvino at kcl.ac.uk (Ruben Calvino) wrote in message news:<1c8875d6.0112100700.1bc3433f at posting.google.com>...
> Dear all,
> I'm just new to this world of transfecting mammalian cells and not
> very successful at the moment. I'm trying to transfect an
> EBV-transform B cell line (Priess) with just the empty vector pcDNA3.1
> before trying with the gene I'm interested in. I just started with the
> Ca-phosphate method, because of its apparent simplicity, but the cells
> are dying just 3-4 hours after adding the precipitated DNA, and the
> medium (RPMI 1640) becomes very cloudy. This is the protocol I follow:
> DNA precipitation. DNA (15 ug) in 220 ul of water. Add 250 ul 2xHBSS.
> Vortex. Add slowly 31 ul 2M CaCl2. Leave for 30 min. The solution
> becomes cloudy after that.
> CELL TRANSFECTION. 3x106 cells in 5 ml of RPMI + 10% FCS in a 6-well
> plate. I add the 500 ul of DNA and incubate the cells at 37oC.
> RESULTS. Cells start to die after 3-4 hours. Completely dead after 16
> hours. Cell culture media very cloudy after just 3-4 hours.
> Any suggestion? What is going on here? I just tried to spin any
> remaining cell alive and resuspended in fresh media but during the
> spin all that precipitate also goes to the botton of the tube and so
> far it doesn't look to improve cell survival.
> Any suggestion welcome
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