COS-7 Cell Electroporation

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Jul 16 20:50:23 EST 2001


lhom at OCF.Berkeley.EDU (Louis Hom) wrote:
:I'm new to electroporating mammalian cells and I'd appreciate any tips on
:what parameters I should pay attention to most for transient expression
:(e.g., preincubation, electroporation medium, recovery medium).  I 
:apologize for such a broad request, but I read a collection of user 
:surveys on Bio-Rad's site and they all seem to say different things.  

Well, since you did not like a multitude of opinions, here is the 
one and the ultimate. Disregard everything else :-)))) 

The most important parameters are pulsing. Use longest pulse 
available to you: Biggest capacitor with no shunting resistance, 
widest gap in cuvettes (probably won't find anything   above 0.4 
cm), and smallest volume (0.4-0.5 ml rathen than 1.0 ml many 
people employ). With this and your choices from below, "titrate" 
voltage to find your optimum. (Make sure to include DNA at 
working concentration, for electroporative cell death is partially 
DNA-dependent). 

Medium choice primarily affects viability (higher survival 
allows to increase V while maintaning the same %death, 
and higher V means higher efficiency of transfection). 
Best results are obtained with media imitating cytosol 
composition - high K, low Na. Good buffering is a plus. 
Something like 30 mM Hepes, 135 mM KCl, 5 mM NaCl,
2 mM MgCl2, pH 7.5 (I suspect using KGlutamate or KGluconate
might be a lot better, but I've never bothered to check; cells 
generally don't like high Cl-; also, large anion-based medium will 
have lower conductivity, which gives you larger tau). Sterilize
by filtration. (I recall seeing some paper, which I have not 
bothered to file, which "discovers' that solution recipie designed 
for storage of organs for transplanation works best in 
electroporation; little wonder then - it's exactly what I describe 
above). 
        Optional but cetainly helps: make 500 mM ATP, 200 mM 
glutathione stock, store at -20, use as 100X to add to you 
cell suspension before electroporation. ATP helps cells to recovery
and GI protects somewhat from massive oxidative damage. 

Final cell concentration 2-4e7/ml (typically one 70% confluent 10 cm
per single point). Efficiency rises with [cells].

Plasmid. Use as much as you are not greedy to use. Efficiency rises
~ linearly with [DNA] upt to ~ 100 ug/ml. 10-20 ug/point is most 
commonly used. Very pure DNA works best (Cesium or 
DEAE-based kits), but for quick and dirty experiments one 
silica-based miniprep/point works just fine too. 

Room temperature works best. No preincubation of . cells with 
DNA is necessary. Postincubation is optional. I usually let them
sit for ~ 5 min in the cuvette, then transfer and let reseal at rt
for ~ 15 min before moving at +37C (warm medium during 
resealing stage somehow  decreases viability). 

That's about all there is. As with everything else, the important 
thing is to understand what exactly you are doing and what every 
parameter does. 

Hope it helps you to decide on what works best for you. 

        - Dima





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