A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia
Data(s) |
30/09/2007
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Resumo |
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.<br /> |
Identificador | |
Idioma(s) |
eng |
Publicador |
Elsevier BV |
Relação |
http://dx.doi.org/10.1016/j.jneumeth.2007.06.009 |
Direitos |
2007, Elsevier B.V. |
Palavras-Chave | #acridine orange #astrocyte #microglia #lysosome #fluorescence microscopy #cell counting #cell culture |
Tipo |
Journal Article |