AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells.
Eur J Histochem
; 56(4): e44, 2012 Oct 11.
Article
em En
| MEDLINE
| ID: mdl-23361240
ABSTRACT
An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Astrocitoma
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Autofagia
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Software
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Perfilação da Expressão Gênica
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Proteínas Associadas aos Microtúbulos
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article