Lipid Rafts. Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues
	1 Introduction
	2 Materials
		2.1 Preparation of Detergent-Resistant Membrane Fractions
		2.2 Sucrose Density Gradient Centrifugation
	3 Methods
		3.1 Preparation of Detergent-Resistant Membrane Fractions
		3.2 Sucrose Density Centrifugation of Detergent-Resistant Membranes
		3.3 General Considerations on Experimental Conditions for Lipid Raft Isolation and Analysis
			3.3.1 Background
			3.3.2 Temperature
			3.3.3 Type of Detergent
			3.3.4 Protein-Detergent Ratio
			3.3.5 Detergent-Free Methods
			3.3.6 DRM from Tissues
			3.3.7 Analysis of DRM Fraction: Importance of Lipid Analysis
			3.3.8 Immunoseparation of DRM Complexes
			3.3.9 Conclusions
	4 Notes
	References
Chapter 2: A Detergent-Free Method for Preparation of Lipid Rafts for the Shotgun Lipidomics Study
	1 Introduction
	2 Materials
		2.1 Plasma Membrane
		2.2 Lipid Rafts
		2.3 Shotgun Lipidomics
	3 Methods
		3.1 Plasma Membrane
		3.2 Lipid Rafts
		3.3 Lipid Extraction
		3.4 Mass Spectrometric Analysis
	4 Notes
	References
Chapter 3: Biochemical Analysis of Lipid Rafts to Study Pathogenic Mechanisms of Neural Diseases
	1 Introduction
	2 Materials
		2.1 Lysate Preparation
		2.2 Sucrose Gradient
		2.3 Immunoblotting
	3 Methods
		3.1 LR Preparation
		3.2 Quality Control of LR Preparation
		3.3 LR Analyses
	4 Notes
	References
Chapter 4: Amyloid-β Interactions with Lipid Rafts in Biomimetic Systems: A Review of Laboratory Methods
	1 Introduction
		1.1 From Micrometer- to Nanometer-Scale Rafts in Biomimetic Systems: Molecular Packing, Lo/Ld Demixing Temperature, Domain Dyn...
		1.2 Amyloid-β Interactions with Raft-like Domains, Dynamics and Size Distributions
	2 Materials
		2.1 Peptides
		2.2 Lipids
	3 Methods
		3.1 Formatin of Lo/Ld Phase Separated Large Unilamellar Vesicle (LUVs)
		3.2 Formation of Lo/Ld Phase Separating Giant Unilamellar Vesicle (GUVs)
		3.3 Aβ (1-42) Aggregation Conditions and Characterization: A Key Problem in Alzheimer's disease research
			3.3.1 Aliquoting Aβ-Stock Film Preparation
			3.3.2 Aβ (1-42) Aqueous Solubilization. Aβ Oligomer Preparation, and Incubation in Vesicle Suspensions
			3.3.3 Determination of the Aβ Concentration Before and After Filtration
				Obtain the Standard Curves for Calibration of Protein Content, Optical Density Approach
				Estimation of Unknown Aβ Content Using the Standard Curves
			3.3.4 Aβ Kinetics Thioflavin T Measurements
			3.3.5 Dynamic Light Scattering (DLS)
			3.3.6 Transmission Electron Microscopy (TEM)
		3.4 Fluorescence Measurements of Lo/Ld Phase Separated  GUVs
			3.4.1 Set Up Needed for Fluorescence Microscopy of Lo/Ld Phase Separated  GUVs
			3.4.2 Choice of Lipid Fluorophore for the Visualisation Lo/Ld Phase Separation
			3.4.3 Studies on Native Domains, Avoiding Photosensitizing Effect of the Lipid Fluorophore, to Define the Demixing Temperature...
			3.4.4 Determination of the Lo/Ld Demixing Temperature
			3.4.5 The Application of Photogenerated Domains in GUVs to Study the Lo/Ld Phase Spinodal Decomposition and Its Modulation by ...
				Fluorescence Imaging Parameters to Observe Photogenerated Domains in  GUVs
				How to Photogenerate Domains in GUVs?
				Photogeneration of Domains in GUVs by Mechanism of Spinoidal Decomposition
				Parameters Determining the Mechanism of Domain Photogeneration
				Conditions to Determine Properly the Kinetics of Photogenerated Domains in  GUVs
				Kinetics of the Appearance and Formation of Photogenerated Domains in GUVs and Size Distribution Determination of the Domains
				Image Processing and Quantitative Analysis
		3.5 Spectroscopic Measurements to Assess the Membrane Lipid Packing and Raft-like Nanoscale Domain Formation
			3.5.1 Membrane Packing Measurements
			3.5.2 Determination of Nanoscale Lo/Ld Demixing Temperature
	4 Notes
	References
Chapter 5: Extracellular Vesicles Containing Ceramide-Rich Platforms: ``Mobile Raft´´ Isolation and Analysis
	1 Introduction
	2 Materials
		2.1 Mice
		2.2 Serum Collection
		2.3 Exosome Isolation
		2.4 Antibody and Affinity Beads
		2.5 Nano Particle Tracking
		2.6 Immunoblotting (Western and Dot Blots)
		2.7 ELISA
		2.8 Mass Spectrometry
		2.9 Recipient Cells
		2.10 Labeling of EVs with Fluorescent  Dye
	3 Methods
		3.1 Preparation of EVs from Serum Using ExoQuick
		3.2 Characterization of Exosome Size and Concentration
		3.3 Affinity Purification Using Ceramide Beads
		3.4 Immunoblotting (Western Blot and Dot Blot)
			3.4.1 Western  Blot
			3.4.2 Dot  Blot
		3.5 ELISA
		3.6 Exosome Labeling and Cell Uptake
			3.6.1 Exosome Labeling with PKH67
			3.6.2 Cultured Cells
		3.7 Mass Spectrometry Analysis
	4 Notes
	References
Chapter 6: Isolation of Lipid Rafts (Detergent-Resistant Microdomains) and Comparison to Extracellular Vesicles (Exosomes)
	1 Introduction
	2 Materials
		2.1 MES
		2.2 Gradient Ultracentrifugation
	3 Methods
		3.1 Isolating Lipid Rafts from Monolayer Cultured Cells
		3.2 Extraction of LRs
		3.3 Sucrose Density Gradient Fractionation
		3.4 Identifying Lipids in the LR Fraction
		3.5 Isolating LRs from Mouse Brain and Other Tissues
		3.6 Sucrose Density Gradient Fractionation
		3.7 Identifying the LR Fraction
		3.8 Nondetergent Methods for Isolating LRs
	4 Notes
	5 General Considerations When Comparing LRs and EVs
	6 Summary
	References
Chapter 7: Methods to Characterize Synthesis and Degradation of Sphingomyelin at the Plasma Membrane and Its Impact on Lipid R...
	1 Introduction
	2 A: Synthesis of SM at the Plasma Membrane
		2.1 Methods
			2.1.1 Method A1: Assay of Sphingomyelin Synthase Activity Using NBD-Ceramide as a Substrate
				Biological Samples
				Lipids
				Buffers
				Mobile Phases
			2.1.2 Procedures
				Step 1: Preparation of the Sample-Substrate  Mix
				Step 2: Incubation for Enzymatic Activity Essay
				Step 3: Measuring the Formation of NBD-SM from NBD-Ceramide
			2.1.3 Method A2: Assay of SMS Activity Using Radiolabeled Phosphatidyl Choline (PC) as Substrate
				Biological Samples
				Lipids
				Buffers
				Mobile Phases
				Procedures
					Step 1: Delivery of the Substrate to the Membrane
					Step 2: Enzymatic Activity Incubation and Product Formation Detection
			2.1.4 Method A3: Assay of SMS Activity Using Radiolabeled Ceramide as Substrate
				Biological Samples
				Lipids
				Buffers
				Procedures
					Step 1: Delivery of the Substrate to the Membrane
					Step 2: Enzymatic Activity Incubation and Product Formation Detection
		2.2 Notes
	3 B: Turnover of SM to Ceramide
		3.1 Method B1: Assay of Neutral Sphingomyelinase-2 in Isolated Plasma Membrane Preparations Using NBD-SM as a Substrate
			3.1.1 Biological Samples
			3.1.2 Lipids
			3.1.3 Buffers
			3.1.4 Mobile Phases
			Procedures
				Step 1: Preparation of Master Mix (for 1 ml, Sufficient for 40 Samples)
				Step 2: Enzymatic Activity Incubation and Product Formation Detection
		3.2 Method B2: Assay of Neutral Sphingomyelinase-2 in Isolated Plasma Membrane Preparations Using Radiolabeled Substrate ([N-M...
			3.2.1 Reagents and Solutions
				Procedures
				Step 1: Preparation of Liposomes of Substrate
				Step 2: Enzymatic Activity Incubation and Product Formation Detection
		3.3 Method B3: Assay of Secretory Sphingomyelinase Activity
			3.3.1 Reagents and Solutions
				Biological Samples
				Lipids
				Reagents
				Procedures
					Step 1: Prepare 0.5 M Acetic Buffer
					Step 2: Prepare Two Master Mixes: With Zinc and Without Zinc (with EDTA) (for 15 Reaction)
					Step 3: Enzymatic Reaction and Product Detection
		3.4 Method B4: Assay of Lysosomal SMase Activity
		3.5 Notes
	References
Chapter 8: Association of Glycolipids and Growth Factor Receptors in Lipid Rafts
	1 Introduction
	2 Materials
		2.1 Buffers
			2.1.1 Base Buffer
			2.1.2 Cell Lysis Buffer
			2.1.3 6x Sample Buffer
			2.1.4 10x Tris-Glycine Buffer
			2.1.5 1x SDS Running Buffer
			2.1.6 1x Transfer Buffer
		2.2 Preparation of Coated Coverslips and Reagents for Confocal Microscopy
			2.2.1 Reagents and Materials
			2.2.2 Reagent Preparation
			2.2.3 Preparation of Poly-l-Ornithine- and Fibronectin-Coated Coverslips
		2.3 Cell Culture
		2.4 Density Gradient Preparation
			2.4.1 Equipment and Supplies
	3 Methods
		3.1 Colocalization of Glycolipid and Growth Factor Receptor Using Confocal Microscopy
			3.1.1 Preparation of Poly-l-Ornithine- and Fibronectin-Coated Coverslip
			3.1.2 Preparation of NSCs Treated with Different Concentrations of  EGF
			3.1.3 Preparation and Fixation of Cells on Coverslips
			3.1.4 Fluorescent Immunocytochemistry Staining of NSCs on Coverslips
			3.1.5 Confocal Microscopy: Expected Results and Data Analysis (See Note 1)
		3.2 Fractionation of Lipid Raft-Associated Growth Factor Receptors Using a Detergent-Free Density Gradient Method
			3.2.1 Cell Membrane Preparation from NSCs for Isolation of Lipid Rafts (See Note 3)
			3.2.2 Preparation of OptiPrep Density Gradients and Fractionation of a Gradient
			3.2.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting
			3.2.4 Expected Results and Data Interpretation
		3.3 Interaction of Glycolipids with Growth Factor Receptors Using Coimmunoprecipitation (Co-IP)
			3.3.1 Cell Lysates Preparation and Immunocomplex Formation
			3.3.2 Magnetic Beads Prewash
			3.3.3 Immunoprecipitation
			3.3.4 SDS-PAGE, Western Blotting and Expected Results
	4 Notes
	References
Chapter 9: Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence ...
	1 Introduction
	2 Materials and Equipment
		2.1 Lipid Rafts Isolation Through Sucrose Gradient Centrifugation
			2.1.1 Buffers and Solutions
			2.1.2 Materials
			2.1.3 Equipment (Other than Generic Laboratory Equipment) (See Fig. 1)
		2.2 Western Blot Analysis for the Presence of Fas/CD95 in Lipid Raft Fractions
			2.2.1 Buffers and Solutions
			2.2.2 Reagents
			2.2.3 Materials
			2.2.4 Equipment (See Fig. 1)
		2.3 Visualization of Fas/CD95 Recruitment in Lipid Rafts by Confocal Microscopy
			2.3.1 Buffers and Solutions
			2.3.2 Reagents
			2.3.3 Materials (See Fig. 2)
			2.3.4 Equipment (See Fig. 2)
	3 Methods
		3.1 Lipid Raft Isolation by Sucrose Gradient Centrifugation
		3.2 Western Blot Analysis of Sucrose Gradient Fractions
		3.3 Visualization of Fas/CD95 Recruitment in Lipid Rafts by Fluorescence Microscopy
			3.3.1 Cell Attachment and Fixation
				Suspension Cells
				Staining
				Adherent Cells
	4 Notes
	References
Chapter 10: Elucidating the Role of Lipid Rafts on G Protein-Coupled Receptor Function in the Mouse Kidney: An In Vivo Approach
	1 Introduction
	2 Materials
		2.1 Animals and Animal Husbandry
		2.2 Preparation of Reagents and Mini Osmotic  Pump
		2.3 Anesthesia, Analgesia, and Surgical Preparation
		2.4 Laparotomy and Implantation of Mini Osmotic  Pump
		2.5 Wound Closure and Recovery
		2.6 Post-surgical Analgesia and Monitoring
		2.7 Euthanasia and Tissue Fixation
		2.8 Verification of Lipid Raft Integrity
		2.9 Other Considerations
			2.9.1 Ventilation
			2.9.2 Femoral Artery/Vein Catheterization
			2.9.3 Urinary Bladder Catheterization (Nonsurvival Surgery)
			2.9.4 Preparation of Surgical Instruments
			2.9.5 Use of Metabolic Cages
	3 Methods
		3.1 Animals and Animal Husbandry
		3.2 Preparation of Reagents and Mini Osmotic  Pump
		3.3 Anesthesia, Analgesia, and Surgical Preparation
		3.4 Laparotomy and Implantation of Mini Osmotic  Pump
		3.5 Wound Closure and Recovery
		3.6 Post-surgical Monitoring
		3.7 Euthanasia
		3.8 Verification of Lipid Raft Integrity
		3.9 Other Considerations
			3.9.1 Ventilation
			3.9.2 Femoral Artery/Vein Catheterization
			3.9.3 Urinary Bladder Catheterization (Nonsurvival Surgery)
			3.9.4 Preparation of Surgical Instruments
			3.9.5 Use of Metabolic Cages
	4 Notes
	References
Chapter 11: Analysis of Lipids in Ceramide-Enriched Membrane Domains
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Immunolabeling
		2.3 Radiolabeling of Surface Ceramide and Sphingosine
	3 Methods
		3.1 Immunochemical Analysis of Ceramide Within Ceramide-Enriched Membrane Domains (See also Fig. 1)
		3.2 Enzymatic Assays to Measure Ceramide and Sphingosine on Cell Surfaces In Situ (Fig. 2)
	4 Notes
	References
Chapter 12: Chemotherapeutic Agents-Induced Ceramide-Rich Platforms (CRPs) in Endothelial Cells and Their Modulation
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Ceramide-rich Microdomains/Platform Detection by Confocal Microscopy
	3 Methods
		3.1 Cell Culture
		3.2 Ceramide-rich Microdomains/Platform Detection by Confocal Microscopy
	4 Notes
	References
Chapter 13: Biophysical Analysis of Lipid Domains by Fluorescence Microscopy
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Probes Stock Solutions
		2.3 Studies with Model Membrane Systems
			2.3.1 Material
			2.3.2 Preparation of Lipids Stock Solutions
			2.3.3 Buffers
			2.3.4 Other Reagents
		2.4 Studies with Cells
			2.4.1 General Material
			2.4.2 Cell Culture
			2.4.3 Other Reagents
	3 Methods
		3.1 Influence of GlcCer in Raft-Domain Formation, Organization and Properties: Studies in Model Membranes
			3.1.1 Methods to Enhance GUV Adhesion/Precipitation to the Bottom of the Microscopy Chamber
				Strategy A: Sucrose-Glucose Density Gradient
				Strategy B: Biotin/Avidin
		3.2 Preparation of GUV by Electroformation
			3.2.1 Option A: With Platinum Electrodes
				A3. Cleaning the Electrodes
			3.2.2 Option B: With Titanium Slides
		3.3 Domain Visualization and Analysis by Confocal Microscopy
		3.4 Microscopy Analysis of the Impact of GlcCer in Living Cell Membranes
			3.4.1 Cell Culture
			3.4.2 Cell Staining
			3.4.3 Cell Imaging and Analysis
	4 Notes
	References
Chapter 14: Biophysical Analysis of Lipid Domains in Mammalian and Yeast Membranes by Fluorescence Spectroscopy
	1 Introduction
	2 Materials
		2.1 Fluorescence Spectroscopy
			2.1.1 Equipment
			2.1.2 Buffers
			2.1.3 Fluorescent Probes (See Notes 4 and 5)
		2.2 Studies in Raft-Mimicking Mammalian Model Membranes
			2.2.1 Lipid Stock Solutions (See Notes 4 and 6)
			2.2.2 Phenolic Compounds Stock Solutions
		2.3 Preparation of Yeast Cell Cultures
			2.3.1 Solutions
			2.3.2 Liquid and Solid Media
	3 Methods
		3.1 Preparation of Raft-Mimicking Large Unilamellar Vesicles
		3.2 Steady-State Fluorescence Anisotropy to Characterize the Fluidity of Lipid Raft Domains
		3.3 Characterization of Lipid Raft Domains by Time-Resolved Fluorescence Spectroscopy
		3.4 Application of FRET to Study Membrane Domains Remodeling
			3.4.1 Incubation with Phenolic Compounds
		3.5 Evaluating the Effects of Phenolic Compounds in the Model Membranes
			3.5.1 Hetero-FRET
			3.5.2 Homo-FRET
		3.6 Characterization of Lipid Domains and Rafts in Fungi Through Fluorescence Spectroscopy
			3.6.1 Culture Preparation
			3.6.2 Cell Preparation
			3.6.3 Fluorescence Assays with Fungal Cells
	4 Notes
	References
Chapter 15: Characterization of Lipid Order and Domain Formation in Model Membranes Using Fluorescence Microscopy and Spectros...
	1 Introduction
	2 Materials
		2.1 Fluorescence Anisotropy
			2.1.1 Reagents
			2.1.2 Equipment
		2.2 FRET
			2.2.1 Reagents
			2.2.2 Equipment
		2.3 GUV Imaging
			2.3.1 Reagents
			2.3.2 Equipment
		2.4 Leakage Assay
			2.4.1 Reagents
			2.4.2 Equipment
	3 Methods
		3.1 Fluorescence Anisotropy
		3.2 FRET
		3.3 GUV Imaging
		3.4 Leakage Assay
	4 Notes
	References
Chapter 16: Characterization of the Effect of Sphingolipid Accumulation on Membrane Compactness, Dipole Potential, and Mobilit...
	1 Introduction
	2 Materials
		2.1 Reagents for Cell Culturing and for Generating Macrophages with and without the Gaucher Phenotype
		2.2 Reagents for FRAP Measurements
		2.3 Reagents for Dipole Potential Measurements
		2.4 Reagents for Measuring Fluorescence Anisotropy and Generalized Polarization
	3 Methods
		3.1 Fluorescence Recovery After Photobleaching
			3.1.1 Preparation of THP-1 Cells for FRAP Analysis of Lipids
			3.1.2 Transfection of THP-1 Cells for FRAP Analysis of Proteins
			3.1.3 Final Preparation of Samples for FRAP Analysis of Lipid Mobility
			3.1.4 Final Preparation of Samples for FRAP Analysis of the Mobility of Ectopically Expressed Proteins
			3.1.5 Microscopy
			3.1.6 Evaluation of the FRAP Experiment
		3.2 Measurement of the Dipole Potential
			3.2.1 Preparations for Measurement of the Dipole Potential
			3.2.2 Microscopy
			3.2.3 Evaluation of Images for Dipole Potential Measurements
		3.3 Measuring Fluorescence Anisotropy and Generalized Polarization of Laurdan by Fluorimetry
	4 Notes
	References
Chapter 17: Superresolution Microscopy of Sphingolipids
	1 Introduction
	2 Materials
		2.1 Antibodies and Toxins
		2.2 Amine Reactive  Dyes
		2.3 Antibody and Toxin Coupling
		2.4 Staining and Superresolution Microscopy by dSTORM
		2.5 Microscopy Setup
	3 Methods
		3.1 Antibody and Toxin Coupling
		3.2 Sample Preparation and Labeling
		3.3 Data Acquisition and Analysis
	4 Notes
	References
Chapter 18: Detection of Functionalized Sphingolipid Analogs in Detergent-Resistant Membranes of Immune Cells
	1 Introduction
	2 Materials
		2.1 Functionalized Ceramide Analog and Cell Culture
		2.2 Isolation of Detergent Resistant Membranes (DRMs)
		2.3 SDS-Polyacrylamide  Gel
		2.4 Semidry Western Blotting
		2.5 Antibodies for Immunoblotting
		2.6 Equipment
	3 Methods
		3.1 T-Cell Labeling with Functionalized C16 Ceramide
		3.2 Validation of Ceramide Specific Click Reaction Efficiency by Flow Cytometry
		3.3 Analysis of Incorporation of Functionalized Ceramide in Cellular Membranes
		3.4 TCR Stimulation by α-CD3 Antibody-Mediated Cross-Linking
		3.5 Isolation of  DRMs
		3.6 Quantification of Fluorescent Ceramide in DRM Fractions
		3.7 10% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
		3.8 Electrophoretic Transfer
		3.9 Western Blot Incubation with the Antibodies
	4 Notes
	References
Chapter 19: Immunofluorescence Labeling of Lipid-Binding Proteins CERTs to Monitor Lipid Raft Dynamics
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Transfection
		2.3 Labeling
		2.4 Microscopy
	3 Methods
		3.1 Cell Culture
		3.2 Transfection
		3.3 Fixation and Blocking
		3.4 Labeling with Cholera Toxin Subunit B (See Note 5 and 6)
		3.5 Labeling with CERTL mAb1 Antibody (See Note 8)
		3.6 Labeling with CERTs HPA608 Antibody
		3.7 Labeling with Secondary Antibodies-Donkey Anti-mouse 594 and Donkey Anti-rabbit 647
		3.8 Mounting and Imaging
		3.9 Image Analysis
	4 Notes
	References
Chapter 20: Cross-Link/Proximity Ligation Assay for Visualization of Lipid and Protein Complexes in Lipid Rafts
	1 Introduction
	2 Materials and Equipment
		2.1 Cell Culture and Cross-linking of Photoactivatable Ceramide (pacFACer)
		2.2 Tagging with Fluorophore or Biotin Using Click Chemistry
		2.3 Pull-down with Streptavidin Agarose or Magnetic Beads
		2.4 Proximity Ligation Assay
		2.5 Immunocytochemistry
	3 Methods
		3.1 Cross-Linking of Photoactivatable Ceramide (pacFACer)
		3.2 Tagging with Fluorophore or Biotin Using Click Chemistry
		3.3 Pull-down with Streptavidin Agarose or Magnetic Beads
		3.4 Proximity Ligation Assay and Immunocytochemistry
	4 Notes
	References
Index