I have seen this before where the reagent remains in the bathing solution.
Can you remove the dye from the cells?
Alternatively, can you fill the sample line with dye at the same
concentration as you've used for the test labelling, prior to each running
of the cells that are in test.
Hope this helps.
Bob
-----Original Message-----
From: Geoffrey Osborne [SMTP:Geoff.Osborne@anu.edu.au]
Sent: Tuesday, August 05, 1997 10:12 AM
To: Cytometry Mailing List
Subject: NBd signal drift: Help?
Hello,
I'm after some advice on a phenomenon I have seen occuring in a
users experiments with the Molecular Probes dye NBD Ro-1986 (cat # N-3413)
which is benzodiazepine coupled and selective for the GABAa receptor.
The problem: Mean fluorescence intensity drifts upwards from around
80 relative fluorescence intensity units on a log scale to around 180 or
higher within a 10 to 15 second time span before gradually stabilising, i.e
continues to drift upwards very slightly. If the cells are removed from the
instrument and then placed on it again the same thing happens. Negative
control remains negative, i.e. mean fluor doesn't increase, no fluorescence
carry over if run through the instrument after a positive sample. Yesterday
when I experienced this, for the second time, the instrument had been
sorting unstained cells for 6 hours so I doubt there was carryover of dye
from a previous experiment.
Conditions of experiment: Cells are SF9 insect cell line. Varying
times post infection. Cells are suspended in PBS, sheath fluid is PBS, both
from the same source. Cell scatters remain constant and characteristic for
infected and non-infected cells. Fluorochrome is being excited at 457nm
with
200mW laser power and emission is collected through a 530/20bp filter.
A similar, though lesser affect, appears to occur at 488nm when the
cells are run on a FACScan.
I'd appreciate any input that anyone has on how/why this "drift"
can occur and if someones got a solution to the problem, well even better.
Thanks in advance,
Geoff
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Geoffrey Osborne | ____ __ o Ahh!
Flow Cytometry (FACS LAB) | __ `\ <,_
John Curtin School of Medical Research, | __ (*)/ (*)
Australian National University, | ==============|
CANBERRA, AUSTRALIA. | |--|
Email: Geoff.Osborne@anu.edu.au | |--|...
Phone: 61 6 249 3694
FAX: 61 6 249 2595
-----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------
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