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To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
Subject: RE: Autofluorescence, Summary
From: Catherine.X.Hu@GSK.COM
Date sent: Fri, 7 Oct 2005 15:11:52 -0400
Dear List Members,
Many thanks to those who respond my questions! The information you
provided are very helpful. I am going through the methods and will give a
try to some of them. Since I may be not the only person having this
issue, I summarize all the responses here and hopefully it will help
anyone out there encountering the similar problem of autofluorescence.
Thank you again,
Catherine
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Original Question:
Dear list members,
For one of my studies, I need to analyze the cytospin slides of cells
fixed with 1% paraformaldehyde using laser scanning cytometer. I found
the autofluorescence of cells getting stronger as time went. The
autofluorescence interfered with the signal of FITC / Alexa Fluor 488 and
made it difficult to get a good separation between negative and positive
populations (usually 24 h after slides being prepared). I could switch to
another dye, but I am trying to develop a multicolor assay and would
rather keep this channel available as long as possible. Has anyone out
there encountered the similar problem? Is there any way to reduce the
autofluorescence? Any input is really appreciated.
Thanks,
Catherine
Catherine Hu
Scientist
Safety Assessment
GlaxoSmithKline
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Response:
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Catherine,
Autofluorescence is usually higher with older aldehyde solutions. I would
recommend using freshly prepared formaldehyde (less than one month old).
Also, the addition of 0.3% Triton X-100 to the antibody incubation
solutions will decrease autofluorescence.
--Randy
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Hi Catherine:
Formaldehydes and glutaraldehydes are bad actors when it comes to
fluorescence-based cytologic analysis. Either get away from fixation with
aldehydes or the answer to the problem is aldehyde blocking. This is done
by reducing the -CHO groups to -OH with sodium borohydride or by
usingbland amino groups (glycine, bovine albumin, skimmed milk).
The other option is to freeze cells. Make cytospins when you need them.
The third option is to live with the autofluorescence. Just dedicate FITC
channel to autofluorescence.
Autofluorescence Eliminator Reagent
http://www.chemicon.com/featured/autofluorescence.asp
http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf
Good luck
Padma
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Hi Catherine,
One method is to use the violet laser for autofluorescence correction.
The blue laser excites both autofluorescence and FITC/Alexa 488.
The violet laser excites only the autofluorescence.
Subtracting the violet excited green scan images from the FITC/Alexa 488
scan images leaves a close approximation to the specific fluorescence.
Best wishes,
Ed
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Dear Catherine,
This is a common problem in immuno-histology.
Attached is a paper that may help.
I have not tried sodium borohydride in a flow setting yet, but one of the
researchers who use my flow lab is going to give it a go.
Regards
Rob W
Message sent using Dodo Internet Webmail Server
[-- Coordinator's note: The paper cited is
Journal of Histochemistry and Cytochemistry, Vol. 49, 1565-1572, December 2001,
Copyright 2001, The Histochemical Society, Inc.
Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue
in Confocal Laser Scanning Microscopy (CLSM)
Werner Baschonga,b, Rosmarie Suetterlin,a, and R. Hubert Laeng,c,
a M. E. Muller Institute at the Biozentrum, Aarau, Switzerland
b Department of Oral Surgery, Radiology and Oral Medicine, Aarau, Switzerland
c University of Basel, Basel, Switzerland, and Department of Pathology,
Kantonsspital, Aarau, Switzerland
Correspondence to: Werner Baschong, M. E. Muller Institute at the Biozentrum,
University of Basel, Klingelbergstrasse 50/70, Basel CH-4056,
Switzerland.
E-mail: Werner.Baschong@unibas.ch
--]
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Hi Catherine,
maybe you can try to fix the slides with 100% methanol for 10 min instead
of PFA. It gives less background but the signal is also less stronger.
Good luck
Simona
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Seems odd for 1 % para fix. are you using the polysciences ultrapure 10 %
stock??
Pb
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Catherine-
You may get a lot of opinions on this, but paraformaldehyde often is a
source of autofluorescence.
(And the longer the cells are exposed to it- the increase in
autofluorescence as days go by).
Years ago I switched to EM grade formaldehyde from Polysciences. It
comes in a box of 10ml ampules, we make it up & only store for a couple
weeks. I fix for min 8 hr in 0.5% formaldehyde. (If you go less, you
may see artifacts in partially fixed cells).
When I need to store fixed cells, I fix for 24hr , then wash &
resuspend in basic FACS buffer (mine is 1X PBS, 0.5% BSA,
0.02%Na-azise). The cells look beautiful for days.
good luck!
b
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if you have a 633 or 635 nm laser may help. At that wavelength
autofluorescence is much less compared to the 488 nm laser.
Ruedi
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Dear Catherine,
Is phenol red a component of the growth medium? If so, I would suggest to
try a medium without phenol red since cells are able to take it up and to
process it further to phenolic compounds which can be the reason for
autofluorescence.
Good luck,
Arne
