PMID- 17395268
OWN - NLM
STAT- MEDLINE
DCOM- 20070815
LR  - 20151119
IS  - 0165-0270 (Print)
IS  - 0165-0270 (Linking)
VI  - 163
IP  - 1
DP  - 2007 Jun 15
TI  - A fluorescence vital assay for the recognition and quantification of excitotoxic 
      cell death by necrosis and apoptosis using confocal microscopy on neurons in 
      culture.
PG  - 1-8
AB  - An automated fluorescence method for the detection of neuronal cell death by 
      necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide 
      (EB) staining using confocal microscopy is described. Since cell nuclei during 
      apoptosis become acidic, AO staining was utilized to distinguish live neurons 
      from neurons undergoing apoptosis, using the AO property to shift its 
      fluorescence from green at normal pH toward brilliant orange-red in the process 
      of acidification. Further EB application labels nuclei of necrotic neurons in 
      red. Sequential treatment by AO and EB can be employed as an express vitality 
      test to count fractions of live and dead cell via apoptosis and necrosis, 
      respectively. An algorithm of automatic quantification of cell types is based on 
      the image correlation analysis. Our conclusion is validated by experiments with 
      the vital dye trypan blue and the pharmacological study of receptor subtypes 
      involved in the excitotoxicity. The approach described here, therefore, offers an 
      express, easy, sensitive and reproducible method by which necrosis and apoptosis 
      can be recognized and quantified in a population of living neurons. Because this 
      assay does not require any preliminary tissue treatment, fixation or dissociation 
      in a cell suspension its utility is likely to be extended for measuring cell 
      viability and cytotoxicity on a variety of living preparations (tissues, brain 
      slices and cell cultures).
FAU - Mironova, Elena V
AU  - Mironova EV
AD  - Sechenov Institute of Evolutionary Physiology and Biochemistry Russian Academy of 
      Sciences, Torez pr. 44, Saint-Petersburg 194223, Russia.
FAU - Evstratova, Alesya A
AU  - Evstratova AA
FAU - Antonov, Sergei M
AU  - Antonov SM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070220
PL  - Netherlands
TA  - J Neurosci Methods
JT  - Journal of neuroscience methods
JID - 7905558
RN  - 0 (Excitatory Amino Acid Agonists)
RN  - 0 (Excitatory Amino Acid Antagonists)
RN  - 0 (Fluorescent Dyes)
RN  - EN464416SI (Ethidium)
RN  - TPY09G7XIR (Fluorescein)
SB  - IM
MH  - Analysis of Variance
MH  - Animals
MH  - Apoptosis/drug effects/*physiology
MH  - Cell Death/drug effects
MH  - Cell Survival/drug effects
MH  - Cells, Cultured
MH  - Cerebral Cortex/cytology
MH  - Drug Interactions
MH  - Embryo, Mammalian
MH  - Ethidium
MH  - Excitatory Amino Acid Agonists/*toxicity
MH  - Excitatory Amino Acid Antagonists/pharmacology
MH  - Female
MH  - *Fluorescein
MH  - Fluorescent Dyes
MH  - Microscopy, Confocal/*methods
MH  - Necrosis/chemically induced/diagnosis
MH  - Neurons/drug effects/*pathology
MH  - Pregnancy
MH  - Rats
MH  - Rats, Wistar
EDAT- 2007/03/31 09:00
MHDA- 2007/08/19 09:00
CRDT- 2007/03/31 09:00
PHST- 2006/07/13 00:00 [received]
PHST- 2007/02/02 00:00 [revised]
PHST- 2007/02/05 00:00 [accepted]
PHST- 2007/03/31 09:00 [pubmed]
PHST- 2007/08/19 09:00 [medline]
PHST- 2007/03/31 09:00 [entrez]
AID - S0165-0270(07)00067-2 [pii]
AID - 10.1016/j.jneumeth.2007.02.010 [doi]
PST - ppublish
SO  - J Neurosci Methods. 2007 Jun 15;163(1):1-8. doi: 10.1016/j.jneumeth.2007.02.010. 
      Epub 2007 Feb 20.