[Cytometry] Acridine orange staining

Darzynkiewicz, Zbigniew Z_DARZYNKIEWICZ at NYMC.EDU
Wed Jul 1 18:46:11 EDT 2009

Acridine orange is not more dangerous dye in terms of contamination of instruments than many other fluorescent dyes (e.g. rhodamine 123). We use AO frequently, rinse the instrument by running with diluted bleach for 5 min and then measure immunofluorescence with no ill effects. If one wants most efficiently clean up the tubing from AO or other fluorochrome I suggest to run the sample containing 0.05M solution of caffeine in PBS. Caffeine has high affinity to form complexes with flat aromatic molecules such as AO or many other dyes and removes them from plastic surfaces or even from already stained cells very effectively by sequestering them in heterologous (caffeine:fluorochrome) complexes(See: Bedner et al. Caffeine dissociates complexes between DNA and intercalating dyes. Application for bleaching fluorochrome-stained cells for their subsequent re-staining and analysis by laser scanning cytometry. Cytometry 2001; 43:38-45.)[Nota bene: caffeine is very inexpensive - I guess it is the by-product of de-caffeinization of coffee and tea]. The myth that AO is irreversibly damaging instruments was developed over 25 years ago when AO was used to measure activation of lymphocytes. Then some reagent companies trying to convince the users to replace this very inexpensive AO test with the assays based on the monoclonal antibody (which costs ~$400) developed the scare tactics presenting AO as the instrument-damaging dye. For some time they even refused to continue warranty of the cytometers if the users were measuring cells stained with AO.

Zbigniew Darzynkiewicz, M.D., Ph.D.

Professor of Pathology and Medicine
Director, Brander Cancer Research Institute
New York Medical College
BSB, Room 438
Valhalla, N.Y. 10595


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Schreiber, Kathy L.
Sent: Wednesday, July 01, 2009 5:36 PM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Acridine orange staining

Hello everyone,

We have a customer at our flow facility who would like to run some
acridine orange-stained samples on the FACScan.  The procedure I have
for cleaning the instrument after running these samples is a 20 minute
rinse with 10% bleach. Is this an adequate cleaning protocol, or can
anyone suggest an alternative that works better?

Thanks in advance for your help,
Kathy L. Schreiber, PhD
Senior Research Specialist
Cell & Immunobiology Core
University of Missouri, Columbia
One Hospital Drive, M324 MSB
Columbia, MO 65212
Phone: (573) 882-3626
Fax: (573) 884-0665
Email: schreiberkl at missouri.edu
<blocked::mailto:schreiberkl at missouri.edu>
Web: www.biotech.missouri.edu/cic
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