Techniques for the Detection of Autophagy in Primary Mammalian Cells.
Autophagy is a lysosomal catabolic pathway responsible for the degradation of cytoplasmic constituents. Autophagy is primarily a survival pathway for recycling cellular material in times of nutrient starvation, and in response to hypoxia, endoplasmic reticulum stress, and other stresses, regulated through the mammalian target of rapamycin pathway. The proteasomal pathway is responsible for degradation of proteins, whereas autophagy can degrade cytoplasmic material in bulk, including whole organelles such as mitochondria (mitophagy), bacteria (xenophagy), or lipids (lipophagy). Although signs of autophagy can be present during cell death, it remains controversial whether autophagy can execute cell death in vivo. Here, we will introduce protocols for detecting autophagy in mammalian primary cells by using western blots, immunofluorescence, immunohistochemistry, flow cytometry, and imaging flow cytometry.
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom;
Daniel
D
Puleston
BRC Translational Immunology Lab, Nuffield Department of Medicine, Oxford University, Oxford OX3 9DS, United Kingdom;
Kanchan
K
Phadwal
BRC Translational Immunology Lab, Nuffield Department of Medicine, Oxford University, Oxford OX3 9DS, United Kingdom;
Alexander Scarth
AS
Watson
Nuffield Department of Clinical Laboratory Sciences, Oxford OX3 9DS, United Kingdom;
Elizabeth J
EJ
Soilleux
The Genome Sciences Centre, BC Cancer Agency, Vancouver V5Z 1L3, Canada;
Suganthi
S
Chittaranjan
The Genome Sciences Centre, BC Cancer Agency, Vancouver V5Z 1L3, Canada;
Svetlana
S
Bortnik
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby V5A 1S6, Canada;
Sharon M
SM
Gorski
Babraham Institute, Cambridge CB22 3AT, United Kingdom;
Nicholas
N
Ktistakis
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; BRC Translational Immunology Lab, Oxford University, Oxford OX3 9DS, United Kingdom.
Anna Katharina
AK
Simon
D000818:Animals; D001343:Autophagy; D002468:Cell Physiological Phenomena; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods*; D006801:Humans; D008322:Mammals