Methods of Adipose Tissue Biology Part B

Contributors
Preface
Chapter One: Preparation and Differentiation of Mesenchymal Stem Cells from Ears of Adult Mice
Abstract

1 Introduction

2 EMSC Isolation, Culture, Cryopreservation, and Differentiation

3 Conclusions

Acknowledgments

Chapter Two: 3-D Adipocyte Differentiation and Peri-adipocyte Collagen Turnover
Abstract

1 Introduction

2 Materials and Methods

Acknowledgment

Chapter Three: Differentiation of White and Brown Adipocytes from Human Pluripotent Stem Cells
Abstract

1 Introduction

2 Human Pluripotent Stem Cells

3 Differentiation into Mesenchymal Progenitor Cells

4 Differentiation into Adipocytes

Chapter Four: Optimal Protocol for the Differentiation and Metabolic Analysis of Human Adipose Stromal Cells
Abstract

1 Introduction

2 Isolation of ASCs from AT

3 Protocols for Proliferation, Subculture, and Freezing Down

4 Protocols to Differentiate Human ASCs to Adipocytes

5 Methods to Use Newly Differentiated Adipocytes for Metabolic Studies

Acknowledgments

Chapter Five: Human Adipose-Derived Stromal/Stem Cell Isolation, Culture, and Osteogenic Differentiation
Abstract

1 Introduction

2 Methods for Cell Culture

3 Methods for Scaffold Preparation

4 Material Characterization

5 Cell Loading and Scaffold Culture

6 Cell Viability and Proliferation

7 Assessing Osteogenic Differentiation

Chapter Six: Analysis of Adipose Tissue Lipid Using Mass Spectrometry
Abstract

1 Introduction

2 Neutral Lipids

3 Phospholipids

4 Summary

Acknowledgments

Chapter Seven: Measurement of Long-Chain Fatty Acid Uptake into Adipocytes
Abstract

1 Introduction

2 Adipocyte Sources

3 Uptake Assay with Radioactive Fatty Acids

4 Uptake Assay with Flow Cytometer

5 Quencher-Based LCFA Uptake Assay

6 Bioluminescent Fatty Acid Uptake Assays

7 In vivo Imaging of BAT Fatty Acid Uptake Rates

Acknowledgments

Chapter Eight: Measurement of the Unfolded Protein Response to Investigate Its Role in Adipogenesis and Obesity
Abstract

1 Introduction

2 Methods to Measure UPR Induction in Adipose Tissues

3 Methods to Investigate Role of UPR in Metabolic Phenotypes of Adipocytes

Acknowledgment

Chapter Nine: Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes
Abstract

1 Introduction

2 Technical Aspects

3 Discussion

Chapter Ten: Measurement of Lipolysis
Abstract

1 Introduction

2 Methodological Description of Activity-Based Assays

3 Conclusion

Acknowledgments

Chapter Eleven: Measurement of Lipolysis Products Secreted by 3T3-L1 Adipocytes Using Microfluidics
Abstract

1 Introduction

2 Methods

3 Discussion

4 Design and Operation Limitations

5 Continued Improvements

6 Notes

Chapter Twelve: Methods for Performing Lipidomics in White Adipose Tissue
Abstract

1 Introduction

2 Tissue Extraction of Adipose Tissue and Adipocytes

3 GC–MS of Total Fatty Acids

4 LC–MS of Intact Lipids

5 Conclusions

Chapter Thirteen: Measuring Respiratory Activity of Adipocytes and Adipose Tissues in Real Time
Abstract

1 Introduction

2 Basis of the Methods and the XF24

3 Cell Culture and Tissue Isolation

4 Preparing the XF24 Analyzer Setup

5 Important Remarks

Acknowledgment

Chapter Fourteen: Detecting Protein Carbonylation in Adipose Tissue and in Cultured Adipocytes
Abstract

1 Introduction

2 Methods to Analyze Protein Carbonylation

3 Results

4 Discussion

Chapter Fifteen: Use of Fluorescence Microscopy to Probe Intracellular Lipolysis
Abstract

1 Introduction

2 Methods to Image and Colocalize Endogenous Lipolytic Effectors in Adipocytes

3 Imaging Protein Trafficking and Protein–Protein Interactions in Live Model Cells

4 Imaging Intracellular Fatty Acid Production

Acknowledgments

Chapter Sixteen: Evaluation of Protein Phosphorylation During Adipogenesis
Abstract

1 Introduction

2 Phosphoproteomic Analysis

3 Western Blot Analysis

4 Phosphorylation Analysis by In Vitro Radiolabeling

5 Enzyme-Linked Immunosorbent Assay

6 Mutation of Phosphorylation Sites

Acknowledgments

Author Index
Subject Index