FACScan FLx drift

Andy Riddell ar3 at mrc-lmb.cam.ac.uk
Thu Nov 28 12:34:02 EST 1996


>From: vanburen%flovax.dnet.wayne.edu at rocdec.roc.wayne.edu
>Date: Wed, 27 Nov 1996 10:20:19 -0500
>To: Cytometry Mailing List <cytometry at 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 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 at 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 control
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 the
cause.

						Hope this helps.




		Andy.........


Andy Riddell
MRC LMB
Hills Road
Cambridge
England
UK
CB2 2QH
email ar3 at mrc-lmb.cam.ac.uk




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