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Pyronin Y Staining to Detect Lymphocyte Activation
Pyronin Y stains double-stranded RNA; it can be excited, albeit
suboptimally, at 488 nm, and emits around 575. In practice, cells stained
with 5 micromolar pyronin Y can be measured in the channel of the flow
cytometer you would normally use for phycoerythrin fluorescence, and pyronin
Y can be used in combination with FITC and with PE-Cy5 and, presumably,
PerCP-labeled antibodies (I don't use PerCP much myself). The dye is pumped
out of live cells by the drug efflux pump; there's usually no problem
staining fixed cells. If you add 1-3 micrograms/ml Hoechst 33342 or 33258,
you can measure DNA as well if you have a dual-beam machine with UV and 488
nm lasers; even when using a single-beam machine, I add the Hoechst dye
anyway on the theory that it blocks any pyronin fluorescence which might
occur from the dye binding to DNA, but that may be more superstition than
science. Reasonably pure Pyronin Y is available in the US from Aldrich and
Polysciences. If you looked at a 2-D DNA vs. RNA (i.e., Hoechst vs. Pyronin
Y) histogram, you'd see something like this:
|
P | SG2M
Y | G1BSSSSSG2M
R R | G1BSSSSSG2M
O | G1BSSSSSG2M
N N | G1AS G2M
I | G1A
A N | G1A
|
Y | G1Q
| G1Q
|_______________________
HOECHST 33342
D N A
The scale is linear (that's important!!!) on both axes; you get the same
picture in cells stained by Darzynkiewicz's method using acridine orange
(AO) if you plot green fluorescence (DNA) on the x-axis and red fluorescence
(RNA) on the Y axis. Resting lymphocytes normally have a G1Q (also known as
G0) RNA content; cells get to G1A about 20 hr after stimulation and, if all
the signals are there, to G1B by around 30 hr. Thereafter, they go through
S, G2, and M and back into G1A, dropping into G1Q when cytokines, nutrients,
and/or stimuli are no longer available. Transformed lymphoblastic cell
lines, e.g., CEM, show the same picture except that there are no G1Q cells.
The advantage of pyronin Y is that it can be used with immunofluorescent
labels excited at 488 nm; with AO, assuming your antigens and/or antibodies
survive the required acid denaturation procedure, you'd need a multibeam
machine and a UV- or red-excited immunofluorescent stain, and the residual
AO in the tubing could screw up your regular immunofluorescence measurements
(FITC, PE, etc.). You have to be careful about washing or bleaching pyronin
out of your tubing as well, but it will interfere in fewer channels, mostly
the PE channel.
In work I did with Bernie Carpenter, Edgar Milford, Terry Strom, and John
Williams about 12 years ago, we found very good correlation between the
number of lymphocytes in a culture with elevated RNA content and the counts
per minute obtained using tritiated thymidine.
I'm happy to answer further questions, at least up to a point.
--Howard Shapiro
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