Front Cover; Molecular Characterization of Autophagic Responses, Part A; Copyright; Contents; Contributors; Preface; 1. Introduction; Acknowledgments; References; Chapter One: Correlative Live Cell and Super Resolution Imaging of Autophagosome Formation; 1. Introduction; 2. Live Cell Imaging; 2.1. Brief Overview of Protocol; 3. Correlative Super Resolution Imaging of Autophagosome Formation; 3.1. Fixation of Samples on the Microscope Stage; 3.2. Labeling of Samples; 3.3. Prepare the Microscope for Structured Illumination Imaging; 3.4. Relocate Cell(s) of Interest

3.5. Acquire 3D Structured Illumination Raw Image Data3.6. Reconstruct 3D Structured Illumination Images and Assess for Artifacts; 3.7. Acquire Raw dSTORM Image Data; Acknowledgments; References; Note added in proof; Chapter Two: Quantifying Autophagic Structures in Mammalian Cells Using Confocal Microscopy; 1. Introduction; 2. Detection and Quantification of Autophagic Puncta in Fixed Mammalian Cells; 2.1. Preparation of Coverslips for Confocal Immunofluorescence Microscopy; 2.1.1. Cell Culture and Autophagy Induction; 2.1.2. Permeablization and Antigen Staining; 2.1.3. Direct Fluorescence

2.1.3. Confocal Imaging2.2. Identifying Autophagic Puncta Using Imaris; 2.2.1. Segmenting Cells, Cytosol, and Nuclei Using Imaris; 3. Quantifying Starvation-Induced ATG9 Redistribution by Indirect Immunofluorescence and Confocal Microscopy; 3.1. Background; 3.2. Cell Culture and Indirect Immunofluorescence Labeling; 3.2.1. Cell Culture and Induction of Autophagy; 3.2.2. Indirect Immunofluorescence Labeling; 3.2.3. Confocal Imaging; 3.3. Image Analysis; 4. Quantification of ATG9 Compartment/Autophagosome Contact in Live Cells; 4.1. Preparation of Cell Cultures for Live Cell Confocal Microscopy

4.2. Analysis of Proximity Between mRFP-ATG9 and GFP-LC3B Structures Using Imaris4.2.1. Manually Creating Cell Surfaces in Imaris; 4.2.2. Measuring Distance Between Two Vesicle Populations Using the Distance Transformation Function; Acknowledgments; References; Chapter Three: The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo; 1. Introduction; 2. Materials; 3. Establishment of Polarized Epithelial Cell Cultures; 4. Incorporation of DQ™-BSA Conjugates; 5. Monitoring Autolysosome Formation; 5.1. Rapamycin Stimulation; 5.2. Serum Starvation

5.3. Evaluation of Autolysosome Formation6. Monitoring LC3-Associated Phagolysosome Formation; 7. Immunofluorescence Analysis; 7.1. Labeling Endogenous LC3; 7.2. Confocal Imaging; 7.3. Important Considerations in Experimental Design; 8. Summary; Acknowledgments; References; Chapter Four: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells; 1. Introduction; 2. Materials; 2.1. Cell Lines; 2.2. Reagents; 2.3. Antibodies; 2.4. Solutions; 2.5. Equipment; 2.6. Labware; 3. Cell Culture, Treatments, and Sample Collection; 3.1. Cell Culture; 3.2. Treatments; 3.3. Sample Preparation