COS-7 Cell Electroporation
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
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
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
Hope it helps you to decide on what works best for you.
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