Transfection of EBV-transformed B cell lines

John Ladasky ladasky at leland.Stanford.EDU
Tue Feb 11 03:54:53 EST 1997


	Cool!  This is starting to get nice and incestuous!  I've met Kiley
Prillman via email, and her boss worked for my PI.  And I read two or three
of Dima Klenchin's articles when I was trying to understand electroporation.

In article <5dm6im$hra at news.doit.wisc.edu>,
Dima Klenchin <klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu> wrote:
>In article <32FE6AE7.7123 at rex.re.uokhsc.edu>, kprillim at rex.re.uokhsc.edu 
>commented on a "magic" problem of transfecting EBV-transformed cells:
>
>#I use essentially the same protocol described by John, but with a few
>#changes I've made (and either with these changes or with dumb luck, I
>#begin to see distinct foci of transfected cells within 2 weeks). =

	I think it also depends rather heavily on the particular cell type
you are growing, and how well you understand its growth conditions.  Getting
the transfected cells to come back from a low-density culture is the real
trick.

>Well, call it my idiot ego, but I can't resist commenting (hope it'll help 
>someone).  
>
>#To begin, I will also give the reference for cytomix, which I agree
>#works well.  The reference is: Nucleic Acids Research, vol. 20, no. 11
>#(van den Hoff et al.).  The "recipe" they give is: 120mM KCl; 0.15mM
>#CaCl2; 10mM K2HPO4/KH2PO4, pH 7.6; 25mM Hepes, pH 7.6; 2mM EGTA, pH 7.6;
>#5mM MgCl2; pH adjusted with KOH.  
>
>Jesus, someone even bothered to publish such a simplistic idea - 
>electroporation medium keeps more cells alive if it resembles an intracellular 
>conditions.

	Well, it *was* a one page technical note in NAR!  No more ink was 
spent on it than required...

>Well, I bet the results will be better if one 1) avoids Ca/EGTA 
>altogether, 2) decreases MgCl2 to 2 mM, 3) adds 0.4% Ficoll 400

	I too have wondered about the Ca++ concentration.  Intracellular Ca++
in resting cells is estimated to be about 100 nM, and high Ca++ generally
implies signal transduction.  Why would you want the cells to signal when
you disrupt them?  It actually sounds like a good way to induce apoptosis!

	Also, I found that my cytomix does *not* keep forever.  After a coup-
le of months, I get a precipitate.  But I autoclaved it instead of sterile
filtering it, and I store it at 4 degrees.   Either or both of these things
may be contributing to my precipitation problem.  I haven't investigated this
any further.

	Could you filter a solution contaning Ficoll?  Or would you have to
autoclave?

>#They suggest that, just prior to use,
>#the cytomix be supplemented with ATP (2mM, pH 7.6 with KOH) and
>#glutathione (5mM), but I do not add these supplements as I have found it
>#unnecessary. 
>
>Exactly. No need at all.

[remainder deleted]

	Regarding electroporation voltages and decay times, and post-elec-
troporation handling, I have to agree with Dima.  And if you look at the 
van den Hoff article, they try different decay times and they concur.  The
longer the pulse, the higher the transfection level -- but more cells die,
so you must find the optimum point.

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green



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