Transfection of EBV-transformed B cell lines
Kiley R. Prilliman
kprillim at rex.re.uokhsc.edu
Sun Feb 9 19:25:23 EST 1997
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).
Incidentally, I also use the Biorad GenePulser apparatus, and I use
either Biorad cuvettes or BTX cuvettes with about equal efficiency
(Biorad ones appear to work just minimally better, though either work
just fine...I believe the BTX cuvettes are cheaper and may be worth it
however if you intend to do a huge number of electroporations).
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. 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. Make up this solution and filter it at 0.2 (it appears to
"keep" FOREVER, so making a large batch is a good thing if you want to
give "consistency" to a series of electroporations). Also, MAKE SURE
you pH it correctly, or it will not work (trust me...I have been through
the pain of being careless with pH-ing!).
I will provide my protocol as follows; I have used it successfully to
electroporate EBV-transformed B-cell lines, other B-cell lines, B-T
fusion hybrid lines, and T-cell lines with either the pBJ1-neo or pcDNA3
(excuse the messiness, I initially wrote this protocol to train some new
individuals in the lab and therefore the grammar is not as great as it
could be!) :)
1) Thaw (if necessary) and culture the cells you plan to transfect and
get them growing nicely in log phase such that they must be cut 1:1
every day (media used here is RPMI-1640 + 10% FCS, always with phenol
red indicator, but you can use whatever medium your cells "like" best:
some B-T fusion lines are happier with IMDM + 10% FCS); the maximum
density I allow them to reach is 600,000 cells/mL. For the two
consecutive days immediately proceeding the actual electroporation,
split the cells with richer media (RPMI-1640 + 20% FCS).
2) On the day of the electroporation, pellet the cells but SAVE the
supernatant drawn from them (this will be used as conditioned media
later on...this appears to be a "key" in getting rapid results).
Resuspend the pellet in 10mL of cytomix and count them. Wash 1x in
cytomix and adjust concentration to 17 million VIABLE cells/mL.
3) Add 750µL of the cell suspension to each electroporation cuvette (be
sure to include cuvettes for "vector-only" and "cells-only" controls
along with your experimental ones!). To this, add 30µg of the DNA (mine
is usually in TE buffer) to be transfected (being certain that the
plasmid volume does not exceed 50µL, as the recommended maximum volume
of the cuvettes is 800µL); to the "cells-only" cuvette, add a similar
volume of TE alone. Mix the DNA with the cells by gently inverting the
capped cuvette a few times.
4) Incubate the cuvettes on ice for 10min. The electroporations are
then carried out at 250V and 960µF capacitance. Just prior to
"zapping", mix the cells once more by gentle inversion and then
electroporate. You may want to watch and/or record the decay time
during this: it should fall between 10-25msec (lower is better).
5) Following each "electrocution", mix the respective cuvette and place
it on ice once again for 10min. Then place the suspensions from the
cuvettes in T-25 tissue culture flasks at a total volume of 6mL
RPMI-1640 + 25% FCS (be sure to get all of the cells, including "globs"
of fried ones, out of the cuvettes and into the flasks by rinsing them
with media). Let these incubate upright at 37°C WITHOUT G418 selection
for 2 days, after which the cultures will appear very yellow.
6) Begin selection on the third day. Count each flask, pellet them, and
then resuspend in selective media (for the "cells-only" flask however,
split it equally and resuspend one half in selective media and the other
half in non-selective media...this provides good visual clues for
determining when "non-transfected cells should be dead"). The selective
medium I use is 1.5mg/mL G418 (I go a little "heavy" I know, but I
prefer it that way and it seems to work just fine) suspended in 1/2
RPMI-1640:1/2 conditioned media (from earlier) + 20% FCS. Be sure to
get the pH correct on this (I use NaOH) and filter sterilize. The
viabilities for the cells counted tend to range from as low as 30% to as
high as 60%. Resuspend them (after pelleting) at a concentration of 1
million viable cells/mL and plate at 2mL/well in a 24-well plate. The
wells should be checked each day to see if they have turned yellow;
generally, the wells will have to be "fed" (consists of gently pulling
off 1mL of media and replacing it with 1mL of fresh selective media) for
two consecutive days following the initiation of selection.
7) Now you "wait and watch": sometimes I have to feed the wells again
for a third day, but not usually. After about 6-7 days some of the
wells may start to appear yellowish; check these out under the
microscope: if tere appears to be "signs of life" (typically by day 4
everything looks like a casualty pile), then wait a few more days (2-3)
and feed the wells in question again. You can kind of play it by ear
after this: after wells are re-fed, the growth tends to speed up as more
transfected cells accumulate. When the well has become almost confluent
in growth, you can begin expanding up to flasks.
Hope this helps somebody,
Kiley R. Prilliman
Department of Microbiology & Immunology
University of Oklahoma Health Sciences Center
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