 |
 |
 |
 |
 |
(1) FL1/FL3 compensation problem.
At the moment, only one investigator is doing 3-color immunofluorescense
experiments (FITC, PE, and Gibco's Red670). These experiments ran fine a
month ago on the "problem" FACScan; now it is impossible to compensate the
Red670 (FL3) signal from the FITC (FL1) channel. Running on another FACScan
resolves this problem. Curiously, at about the same time this problem
started to occur, another investigator had begun using 7-AAD on the same
"problem" FACScan. With this in mind, the "problem" FACScan was cleaned
(30 minutes 10% bleach followed by 30 minutes purified water through both
the sheath and sample lines [sans sheath filter]) ahead of its weekly
schedule. After cleaning, the 3-color experiment appeared to run
appropriately on the "problem" FACScan. A few days later (and after another
7-AAD run), the same problem recurred; this time, a cleaning did NOT
resolve the problem. The same problem can be seen by running CaliBRITE
beads (the standard beads [blank, FITC, and PE] and the PerCP beads). {I
suppose I should be a little more clear on the problem. When FL2-%FL3 and
FL1-%FL2 are adjusted properly for removing FL3 fluorescense from the FL2
signal and FL2 fluorescense from the FL1 signal, the FL3 fluorescense still
appears in the FL1 signal. Overcompensation or either (or both) FL2-%FL3
and FL1-%FL2 will not compensate FL3 from FL1.}
(2) FLx drift problem.
This problem started to occur at about the same time as the other problem,
which makes me wonder if they are somehow related. Independent of
fluorochrome (FITC, PI, 7-AAD) or detector (FL1, FL2, FL3), the fluorescense
signal will drift; that is, over time, the signal usually increases in
intensity. This is most easily seen by running a sample, taking it off the
SIP, putting it right back on the SIP, and re-running it. Even after the
sample pressure boost and a steady sample pressure, the signal will
initially be less intense than it was just moments ago. After several
minutes, the intensity climbs back up. This behaviour has been seen on
intracellular labeling with FITC-conjugated antibodies, and DNA labeling
with either PI or 7-AAD. The samples were not run on another FACScan. The
same problem can be seen by running CTN stained with PI from the DNA QC
kit.
/\/\/\_ Eric Van Buren, vanburen%flovax.dnet@rocdec.roc.wayne.edu
\ \ \ Karmanos Cancer Institute and Immunology & Microbiology,
\_^_/ Wayne State University, Detroit, MI
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web