Transfection by Ca-phosphate of a B-cell line

kalyan kalsriv at mailcity.com
Mon Dec 10 16:49:38 EST 2001


Hi Ruben

Welcome Aboard!

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.

Good Luck.

...Kalyan
 
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
> 
> Regards




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