Re: FACScan FLx drift
Andy Riddell (ar3@mrc-lmb.cam.ac.uk)
Thu, 28 Nov 96 17:34:02 +0000
>From: vanburen%flovax.dnet.wayne.edu@rocdec.roc.wayne.edu
>Date: Wed, 27 Nov 1996 10:20:19 -0500
>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>Subject: FACScan FL1/FL3 comp and FLx drift
>
>
>I am seeking suggestions for remedies to two problems we have been
>experiencing lately on one of our FACScans. This FACScan has had its
>PMI fewer than 6 months ago, and had its laser power supply replaced less
>than a year ago. It was manufactured in June of 1992. These problems did
>not exist prior to a couple weeks ago.
>
>(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 fluorescen=
se
>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
Hi Eric,
I had a similar problem on my FACSCalibur to your FLx drift. It turned =
out
to be one of the hydrophobic air pressure filters behind the fluidics contr=
ol
panel got wet and an 'air lock' of sorts formed. I just replaced the filter=
and
it worked fine. As to how the filter got wet, I haven't yet tracked down th=
e
cause.
Hope this helps.
Andy.........
Andy Riddell
MRC LMB
Hills Road
Cambridge
England
UK
CB2 2QH
email ar3@mrc-lmb.cam.ac.uk
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