Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.
Department of Translational Oncology, Genentech, South San Francisco, California, United States of America.
Michael
M
Degtyarev
Department of Pathology, Genentech, South San Francisco, California, United States of America.
Mike
M
Reichelt
Department of Translational Oncology, Genentech, South San Francisco, California, United States of America.
Kui
K
Lin
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013010:Sonication; D013194:Staining and Labeling; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
C502070:MAP1LC3B protein, human; D018942:Macrolides; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C025946:3-methyladenine; D002738:Chloroquine; C040929:bafilomycin A1; D000225:Adenine