AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells

  • L. Fassina Dipartimento di Ingegneria Industriale e dell’Informazione, University of Pavia, Pavia, Italy; Centro di Ingegneria Tissutale (C.I.T.), University of Pavia, Pavia, Italy, Italy.
  • G. Magenes Dipartimento di Ingegneria Industriale e dell’Informazione, University of Pavia, Pavia, Italy; Centro di Ingegneria Tissutale (C.I.T.), University of Pavia, Pavia, Italy, Italy.
  • A. Inzaghi Centro di Ingegneria Tissutale (C.I.T.), University of Pavia, Pavia, Italy, Italy.
  • S. Palumbo Dipartimento di Biologia e Biotecnologie, University of Pavia, Pavia, Italy, Italy.
  • G. Allavena Dipartimento di Biologia e Biotecnologie, University of Pavia, Pavia, Italy, Italy.
  • C. Miracco Dipartimento di Patologia Umana ed Oncologia, University of Siena, Italy, Italy.
  • L. Pirtoli Dipartimento di Patologia Umana ed Oncologia, University of Siena, Italy, Italy.
  • M. Biggiogera Dipartimento di Biologia e Biotecnologie, University of Pavia, Pavia, Italy, Italy.
  • S. Comincini | sergio.comincini@unipv.it Dipartimento di Biologia e Biotecnologie, University of Pavia, Pavia, Italy, Italy.

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.

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Published
2012-10-11
Section
Original Papers
Keywords:
autophagy, autophagosome, vacuole, GFP, Rapamycin
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How to Cite
Fassina, L., Magenes, G., Inzaghi, A., Palumbo, S., Allavena, G., Miracco, C., Pirtoli, L., Biggiogera, M., & Comincini, S. (2012). AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells. European Journal of Histochemistry, 56(4), e44. https://doi.org/10.4081/ejh.2012.e44