pH changes can cause a phenomenon akin to what you describe. I was running a
series of samples and found that the mean fluorescence increased over time as I
ran the samples just as you describe. Then I noticed that the investigator had
a pH written on each tube. He had a series of tubes at different pHs. I assume
that when the samples were run the pH moved toward neutral because of the
buffering capacity of the sheath fluid, as there will be some mixing. So, you
can check the pH of the samples and adjust them to neutral as long as that
won't mess up your experiment. I suppose you could also just wait until the
signal stops drifting before you start collecting data. Anyone want to comment
on that approach?
David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center
---------------------- Forwarded by David McFarland/VUMC/Vanderbilt on 05/27/99
09:55 AM ---------------------------
Barry Grimes <bagrim1@pop.uky.edu> on 05/25/99 04:33:35 PM
(Embedded image moved to file: pic04580.pcx)From:(Embedded image moved to
file: pic20973.pcx)Barry Grimes <bagrim1@pop.uky.edu> on 05/25/99 04:33 PM
To: Cytometry Mailing List
<cytometry@flowcyt.cyto.purdue.edu>
cc: (bcc: David McFarland/VUMC/Vanderbilt)
Subject: Increases in fluorescence background?
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>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>From: Howard Shapiro <hms@shapirolab.com>
>Subject: Re: 7ADD vs Hoechst
>Date: Sun, 23 May 1999 17:52:09 -0400
snip - Howard Shapiro writes:
>
>At 1 ug/ml, Hoechst 33342 gives good DNA histograms in fixed cells,
>particularly when 0.1% Triton X-100 is added. However, I (and others) have
>observed, but not formally published, that staining of formaldehyde-fixed
>cells with Hoechst dyes and DAPI produces increases in fluorescence
>background in the fluorescein and PE channels. We don't understand the
>mechanism, but believe that the fluorescence is due to a chemical reaction
>between the dye and formaldehyde or some product of formaldehyde. The
>longer the time in fixative, the worse the background; once cells have been
>adequately fixed, adding 0.15% glycine (a suggestion made to us by Richard
>Riese) seems to neutralize the remaining fix and prevent further increases
>in background fluorescence. If I weren't trying to use the formaldehyde to
>neutralize HIV and other nasties in specimens, I'd be tempted to work with
>unfixed or ethanol-fixed cells.
>
>-Howard
>
We have also witnessed that staining of formaldehyde-fixed DAPI produces
increases in fluorescence
background.
This has been observed in experiments using human cells indirectly labeled
with a Red-613 tagged antibody. The cells are permeabilized with tritonX
and then fixed in 1% PF. We are looking at DNA using DAPI.
In these experiments our FL3 channel (Red-613) appears to gain background
fluorescence as we are running the cells through a Vantage. Amazingly, when
the labeled cell suspension is first placed on the sample port, the cells
exhibit normal looking, negative background fluorescence levels then,
within a few seconds, all of the cells begin to show movement into the
positive. The FL1 and FL2 channels do not show this fluorescence creep -
and we cannot reproduce this phenomenon on a FACScan.
It doesn't appear to be sample pressure related as this fluorescence
increase happens in the same manner no matter what the psi of the sample
differential. However, if one takes the cells off the sample port after
they have migrated into the positive and then replaces them again - the
background fluorescence initialy returns to normal negativity - but then
soon begins it's slide towards the positive.
There is a point where the migration into the positive appears to slow and
stop - but it is typicaly a considerable distance from the baseline.
This seems similar to what Howard has described - with the exception that
we witness it in the FL3 channel, but NOT in the FL1 or Fl2. I will be very
interested to see if the addition of the 0.15% glycine helps.
Have any others seen this phenomenon as well?
regards,
barry
<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>:<+>
Barry Grimes
FACS Laboratory Manager
Hematopoiesis Center
Univ. of Kentucky Medical Center
Blood and Marrow Transplant
Rm.cc418 Markey Cancer Center
800 Rose St, Lexington,KY 40536-0093
lab) 606-323-8193
fax) 606-257-7715
e-mail) bagrim1@pop.uky.edu
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