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Monitoring and Measuring Mammalian Autophagy

  • Sujit K. Bhutia
  • Prakash P. Praharaj
  • Chandra S. Bhol
  • Debasna P. Panigrahi
  • Kewal K. Mahapatra
  • Srimanta Patra
  • Sarbari Saha
  • Durgesh N. Das
  • Subhadip Mukhopadhyay
  • Niharika Sinha
  • Prashanta K. Panda
  • Prajna P. Naik
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

Macroautophagy (autophagy) is a conserved lysosomal-based intracellular degradation pathway. Here, we present different methods used for monitoring autophagy at cellular level. The methods involve Atg8/LC3 detection and quantification by Western blot, autophagic flux measurement through Western blot, direct fluorescence microscopy or indirect immunofluorescence, and finally traffic light assay using tf-LC3-II. Monitoring autophagic flux is experimentally challenging but obviously a prerequisite for the proper investigation of the process. These methods are suitable for screening purposes and can be used for measurements in cell lysates as well as in living cells. These assays have proven useful for the identification of genes and small molecules that regulate autophagy in mammalian cells.

Keywords

Autophagic flux LC3-II p62 TEM tf-LC3 

Notes

Acknowledgments

Research support was partly provided by the Department of Biotechnology [Grant Number: BT/PR7791/BRB/10/1187/2013], Board of Research in Nuclear Sciences (BRNS) [Number: 37(1)/14/38/2016-BRNS/37276], Department of Atomic Energy (DAE), and Science and Engineering Research Board (SERB) [Number: EMR/2016/001246]. Research infrastructure was partly provided by Fund for Improvement of S&T Infrastructure in Universities and Higher Educational Institutions (FIST) [Number: SR/FST/LSI-025/2014], Department of Science and Technology, Government of India.

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Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Sujit K. Bhutia
    • 1
  • Prakash P. Praharaj
    • 1
  • Chandra S. Bhol
    • 1
  • Debasna P. Panigrahi
    • 1
  • Kewal K. Mahapatra
    • 1
  • Srimanta Patra
    • 1
  • Sarbari Saha
    • 1
  • Durgesh N. Das
    • 2
  • Subhadip Mukhopadhyay
    • 3
  • Niharika Sinha
    • 4
  • Prashanta K. Panda
    • 5
  • Prajna P. Naik
    • 6
  1. 1.Department of Life ScienceNational Institute of Technology RourkelaRourkelaIndia
  2. 2.Department of MedicineUniversity of Texas Health Science CenterTylerUSA
  3. 3.Department of Radiation OncologyNYU Langone Medical CenterNew YorkUSA
  4. 4.Department of Animal ScienceMichigan State UniversityEast LansingUSA
  5. 5.Institute of Cancer and Genomic Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
  6. 6.PG Department of ZoologyVikram Deb (Auto) CollegeJeyporeIndia

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