A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia

Lovelace, Michael D. and Cahill, David 2007, A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia, Journal of neuroscience methods, vol. 165, pp. 223-229.

Attached Files
Name Description MIMEType Size Downloads

Title A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia
Author(s) Lovelace, Michael D.
Cahill, David
Journal name Journal of neuroscience methods
Volume number 165
Start page 223
End page 229
Publisher Elsevier BV
Place of publication Amsterdam, The Netherlands
Publication date 2007-09-30
ISSN 0165-0270
1872-678X
Keyword(s) acridine orange
astrocyte
microglia
lysosome
fluorescence microscopy
cell counting
cell culture
Summary Cell culture analyses of growth, morphology and apoptosis commonly require counting of different cell types stained with antibodies to discriminate between them. Previously, we reported the use of l-Leucine methyl ester (l-LME) to prepare purified cultures of type 1 astrocytes with minimal microglia, and staining by GFAP and CD antibodies, respectively. Here, we demonstrate a novel use of acridine orange (AO) for rapid discrimination between these cell types using fluorescence microscopy. AO accumulates in the lysosomes and also binds strongly to nuclear DNA and cytoplasmic/nucleolar RNA. Microglia may contain abundant lysosomes due to known roles in homeostasis and immune response. AO staining of lysosomes was tested at a range of concentrations, and 2.5 μg/mL was most suitable. In agreement with previous reports, microglia treated with AO showed very intense yellow, orange or red granular cytoplasmic staining of lysosomes. Microglia contain a substantially higher number of lysosomes than astrocytes, which have a variable amount. We measured the microglia population at 5.14 ± 0.50% in mixed cultures. Thus, these results show AO is a novel discriminatory marker, as microglia were easily observed and counted in clumps on top of the monolayer of astrocytes, providing a rapid alternative to time-consuming and costly antibody-based assays.
Language eng
Field of Research 110703 Autoimmunity
HERDC Research category C1 Refereed article in a scholarly journal
Copyright notice ©2007, Elsevier B.V.
Persistent URL http://hdl.handle.net/10536/DRO/DU:30007743

Document type: Journal Article
Collection: School of Life and Environmental Sciences
Connect to link resolver
 
Unless expressly stated otherwise, the copyright for items in DRO is owned by the author, with all rights reserved.

Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 5 times in TR Web of Science
Scopus Citation Count Cited 8 times in Scopus
Google Scholar Search Google Scholar
Access Statistics: 479 Abstract Views, 0 File Downloads  -  Detailed Statistics
Created: Mon, 29 Sep 2008, 08:55:37 EST

Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.