Question:
Hello netters -
I am working with adherent malignant human epithelial lines
(ovarian and breast cancers), and wish to evaluate a purified population of
transiently transfected cells. The constructs used for transfection are
expected to be growth inhibitory. My best shot at transfection efficiency
is 25% (range 5%-25%) with certain favored cell line/transfection reagent
combos, as evaluated by gfp/fluorescence microscopy. I would like to get a
population of at least 50% transfected, preferably up to 90% transfected
cells for further experiments ON LIVE CELLS, notably growth curves and
other experiments where the readout is greater than 3-4 days post
transfection. 2 X 10e5 live cells are adequate for the output of the
selection process.
Possibilities I am considering are:
1. co-transfection with surface-expressed protein not endogenous to my
cells, positive selection with magnetic beads
2. co-transfection with fluorescent marker and sterile sorting (but the
FACS is not a high-rate instrument)
Does anyone have any experience with these methods, FACS vs. mag.bead
effects on downstream viability, experience with various kits (Miltenyi
MACSelect mouse H-2 Kk kit, Dynabeads with what surface marker). ? I have
slight familiarity with use of beads to fractionate various leukocytes for
immediate analysis.
Answer:
I can't comment on FACS vs magnetic-based separations but there is
one bit I know that should help: you can take TONS of cells (5e7-1e8/ml), mix
them with TONS of DNA plasmid (0.1-0.2 mg/ml final) and electroporate
them under really "harsh" conditions (so do that > 90% of cells will be
seriously dead in ~ 24 hours). Typically, this setup results in nearly all
surviving cells being transfected and expressing your protein/surface
marker. The trick in your case would be to separate dead from alive. FACS
in this case is very straightforward, it is also usually (always?) possible on
density gradients (Ficoll or Percoll), and might be even better with magnetic
separation (although I'd guess with lower yeild). For "better" transfection
and viability, all other things being equal, you'd be better of with as big
capacitance as your equipment allows and with having 2 mM Mg2+ in
the buffer/medium with addition of 5 mM glutatione, 2 mM ATP (GI/ATP
mix is acidic, so it is better to make 100X stock, adjusted pH to ~ neutral
and store frozen).
