PMID- 17662460
OWN - NLM
STAT- MEDLINE
DCOM- 20071120
LR  - 20131121
IS  - 0165-0270 (Print)
IS  - 0165-0270 (Linking)
VI  - 165
IP  - 2
DP  - 2007 Sep 30
TI  - A rapid cell counting method utilising acridine orange as a novel discriminating 
      marker for both cultured astrocytes and microglia.
PG  - 223-9
AB  - 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 microg/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.
FAU - Lovelace, Michael D
AU  - Lovelace MD
AD  - School of Life and Environmental Sciences, Deakin University, Pigdons Road, Waurn 
      Ponds, Victoria 3217, Australia.
FAU - Cahill, David M
AU  - Cahill DM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070617
PL  - Netherlands
TA  - J Neurosci Methods
JT  - Journal of neuroscience methods
JID - 7905558
RN  - 0 (Coloring Agents)
RN  - 0 (Nucleic Acids)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/chemistry/*metabolism/pharmacokinetics
MH  - Animals
MH  - Animals, Newborn
MH  - Astrocytes/chemistry/*cytology/physiology
MH  - Cell Count/methods
MH  - Cells, Cultured
MH  - Central Nervous System/cytology
MH  - Coloring Agents/chemistry/*metabolism/pharmacokinetics
MH  - Female
MH  - Lysosomes/chemistry/metabolism
MH  - Microglia/chemistry/*cytology/physiology
MH  - Microscopy, Fluorescence/methods
MH  - Nucleic Acids/chemistry/metabolism
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Staining and Labeling/*methods
MH  - Time Factors
EDAT- 2007/07/31 09:00
MHDA- 2007/12/06 09:00
CRDT- 2007/07/31 09:00
PHST- 2007/04/23 00:00 [received]
PHST- 2007/06/10 00:00 [revised]
PHST- 2007/06/11 00:00 [accepted]
PHST- 2007/07/31 09:00 [pubmed]
PHST- 2007/12/06 09:00 [medline]
PHST- 2007/07/31 09:00 [entrez]
AID - S0165-0270(07)00277-4 [pii]
AID - 10.1016/j.jneumeth.2007.06.009 [doi]
PST - ppublish
SO  - J Neurosci Methods. 2007 Sep 30;165(2):223-9. doi: 
      10.1016/j.jneumeth.2007.06.009. Epub 2007 Jun 17.