I posted the following message yesterday, however I forgot to mention
that I am using UV excitation of the EMA. That is because I am using up
all of the 488 nm PMTs on our FACS Vantage for 3 other fluorochromes.
The UV laser excites Hoechst 33342 and EMA.
I made up fresh EMA yesterday to compare it to the refrigerated EMA
made on 4/5/00 and tested them on freshly thawed patient cells that were
80-90% viable by trypan blue exclusion versus a cell line that had ~50%
viability. Surprisingly, considering what I saw before, the 5 ug/mL EMA
treatment yielded equivalent results using continuously growing versus
freshly thawed cells. When using 488 excitation, there was a clear
separation between live and dead (or EMA neg and pos, resp.) cells. The
separation when using UV excitation was not as good, but it was there if
one knew what they were looking for. I was also surprised to see that
the EMA that had been sitting in the refrigerator for approx. 3 weeks
gave either the same results as the freshly made EMA or only very
slightly worse. It appears that we did not have the Vantage set up
correctly before.
A question remains, however. Is there a way to get EMA to give stronger
emission when excited with the UV laser. As I said, the difference
between the EMA positive dead cells and the EMA negative live cells is
not as clear when the UV laser is used rather than the 488 laser. I
wouldn't have expected this, because when I used to assay apoptotic DNA
laddering on agarose gels the DNA was stained with ethidium bromide that
was excited with UV light. That fluorescence seemed quite bright to me.
I am sorry that my first message was misleading.
Kevin Waddick
------------------------------------------------------------------------
ORIGINAL MESSAGE
A week ago Howard Shapiro enlightened me (and maybe others) by
saying that, whereas propidium is (supposedly) membrane impermeant to
healthy cells, ethidium is membrane permeant and is pumped out of
healthy cells. Thus, propidium is a viability stain that operates by
passive dye exclusion and ethidium requires active participation on the
part of the healthy cell to appear negative for staining with this dye.
I am wondering if newly thawed cells have the ability to pump out
ethidium -- or, more specifically, ethidium monoazide (EMA) -- if they
are otherwise viable at that point. When I thaw cryopreserved cells I
get a clear-cut idea of their viability immediately afterward by trypan
blue exclusion (I realize that early apoptotic cells are trypan
negative) because it is a yes-or-no decision. When I stain with 5 ug/mL
EMA, leave the cells in the dark for 15 minutes and then expose them to
a nearby fluorescent light source for 15 minutes, I get a confusing
pattern when the cells are analyzed by flow cytometry after washing,
fixing, etc. Not even all of the cells obviously dead by FSC, SSC, and
hypodiploidy are positive for EMA staining. And the seemingly viable
cells have a wide range of EMA positivity, whereby many of them have
higher intensity than the lower intensity dead cells.
So, can EMA even be used right after cells have been thawed, or is
their permeability/dye pumping status totally messed up? I would like to
be able to quickly fix/permeabilize cryopreserved cells after thawing to
prevent further cell death and in preparation for intracellular staining
but be able to distinguish the cells that were dead prior to that point.
Also, is EMA unstable in solution at 4 C or RT, unlike plain old
ethidium bromide? The results I got soon after making a stock solution
of 0.5 mg/mL EMA were quite different from those 2 weeks later when the
stock had been sitting in the refrigerator.
Kevin G. Waddick, Ph.D.
Parker Hughes Institute
2657 Patton Road
St. Paul, MN 55113
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