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Current Protocols in Cell Biology 2010年完整版 5483页

Online ISBN: 9780471143031
DOI: 10.1002/0471143030
- I- h5 E/ J2 R/ j  q9 Y3 r9 {8 g- f5 Q
Table of Contents# S# H3 }$ B- `. |
1. Preface
2. Foreword! p3 R6 l& e9 [) a% O
3. Chapter 1 Cell Culture
1. Introduction3 t' X  Y9 W- G7 K: ~8 K0 T
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
3. Unit 1.2 Media for Culture of Mammalian Cells. c# X' u' e" W5 Z
4. Unit 1.3 Aseptic Technique for Cell Culture9 _/ p5 O, I$ ^) y0 c' _+ }! u; f0 c
5. Unit 1.4 Sterilization and Filtration0 l# b  W5 d/ m& ]+ k; G
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures9 Z# w2 V" O% l5 f: V) ]
7. Unit 1.6 Media and Culture of Yeast- g: d3 y1 h$ \8 F3 B
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging% ?2 u4 W! W3 n5 O/ O, z
4. Chapter 2 Preparation and Isolation of Cells: ]: Z* v2 F3 p8 {1 s$ _
1. Introduction9 U  E! N* w& q7 v3 d2 _8 N1 B+ i
2. Unit 2.1 Establishment of Fibroblast Cultures& N# @( a) w% }: A3 `
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
4. Unit 2.3 Preparation of Endothelial Cells
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation1 r$ n; m- J! D8 J) L
6. Unit 2.5 Laser Capture Microdissection1 e7 Y. F: T& f1 c+ N
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures0 j, u2 n2 ]( c2 @& W" s6 }5 R! G
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
1. Introduction
2. Introduction: z2 m9 {3 {. l* f, Z( n* I
3. Unit 3.1 Overview of Cell Fractionation3 N# e( Q- E% ~9 [0 I
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains% p! b0 m9 \  W
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation) l) L# o% N( H
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation; N5 J$ V+ l5 v- h( i2 Q7 J
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient$ X" Y! b: M4 t
Centrifugation/ }4 G' A8 Q3 M1 U5 B% S& Y: s
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient& K- A4 c* k, ]# P( J5 ~1 e
Centrifugation
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae, h* B( |7 ~0 O& ?
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae1 ^1 J( D. z, v- `
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
Centrifugation% \. `1 {7 ]6 J5 x
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes0 _, D1 L7 ]' j5 C
14. Unit 3.12 Isolation of Synaptic Vesicles2 a6 H3 l" T9 a: J; @# ?
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation4 R/ R+ e9 Q( {( i) K. L) a
16. Unit 3.14 Isolation of Melanosomes
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
18. Unit 3.16 Isolation of Mast Cell Granules! T7 u5 U1 V8 g8 J9 K1 H
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster( d& h9 k: }+ s8 O. k
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas9 R; w% F( x$ e8 C* s; d
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes: Q- P; U+ @. t! i4 q
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological% M9 {  R: u9 r. W
Fluids1 i7 X" Y" i% S! P
25. Unit 3.23 Isolation of Intermediate Filaments" }4 {9 z" U8 j; V" }7 e; b; v8 ]/ l
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle; k9 A4 T+ _" v. \2 ^1 `3 j
27. Unit 3.25 Isolation of Myelin
28. Unit 3.26 Isolation of Renal Brush Borders/ \! s" A; d  }
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane8 u! ^$ L5 Q7 v6 ?( u& L
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts" m$ _' [" o% v$ T; j( ]) z
30. Unit 3.28 Isolation of Amyloplasts$ J4 T6 E6 R% F6 }5 P
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic- n  Y3 A- Z9 e  |6 a, d5 E
Centrifugation
33. Unit 3.31 Isolation of Neuromelanin Granules6 A, ]' f( C, `+ H5 {
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
Density Gradient Centrifugation! h; b! T  U. ^# S' S
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation3 P# p: t0 I8 v4 V+ P$ t
37. Unit 3.35 Isolation of Platelet Granules
38. Unit 3.36 Isolation of Nucleoli, [7 G- w/ V. p
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
41. Unit 3.39 Isolation of Chromaffin Granules
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines$ c8 L& L5 [% b. W- r. s: n* [4 n0 f
6. Chapter 4 Microscopy
1. Introduction
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope0 M7 g& C4 F' u, V
3. Unit 4.2 Fluorescence Microscopy
4. Unit 4.3 Immunofluorescence Staining
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
6. Unit 4.5 Basic Confocal Microscopy% `1 |( o3 L! ?* ?" k& g' q
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
8. Unit 4.7 Cryo-Immunogold Electron Microscopy, b* ?2 G  Q/ v* Z( o
9. Unit 4.8 Correlative Video Light/Electron Microscopy
10. Unit 4.9 Polarization Microscopy5 F5 E: t5 K& U4 k
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells5 o0 M& f+ l3 E+ I) O1 P4 k
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues, C/ D: ~4 Z. ^: J5 W7 F& K
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface8 q# ]" m+ v# m9 }9 ?
Events
14. Unit 4.13 Fluorescent Labeling of Yeast
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy/ h1 l7 t  N$ U( m" _
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules& P$ i2 p8 M" D
in Endocytosis' J! W* [3 i0 f1 Q1 a
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images4 K% l7 A/ A$ s# v: L3 c7 F' _1 a# \
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions/ m/ x) q( F7 N: C1 V
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics, j) W! ~3 X1 [$ g9 X: L
24. Unit 4.23 Interference Reflection Microscopy
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches6 c: c8 f6 N+ _
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
7. Chapter 5 Characterization of Cellular Proteins
1. Introduction
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein0 @/ x4 b; G/ x4 ^4 i
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients: w) [4 r2 z* D9 _
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration); p2 _! D) }. }. \4 X. v
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
Spectrometry
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening2 K1 S- u# n0 s. M
Applications
8. Chapter 6 Electrophoresis and Immunoblotting
1. Introduction
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins3 b* v; D8 P, @7 I0 w
3. Unit 6.2 Immunoblotting and Immunodetection$ x# a: a0 b% P* p
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots- g6 ^( L7 `) g* Q3 x
5. Unit 6.4 Two-Dimensional Gel Electrophoresis$ z% t5 M( N+ G
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions( V" t: V' h1 _& b& c" e% k7 l
7. Unit 6.6 Staining Proteins in Gels
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots' z; W( z, Q  I- x6 n& Q
10. Unit 6.9 Digital Electrophoresis Analysis  t; L' Y* f' [  e3 O9 M0 M5 ^
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
Single Cell Gel Electrophoresis (Comet Assay)
9. Chapter 7 Protein Labeling and Immunoprecipitation  a" ~; l& d8 l3 j# E
1. Introduction
2. Unit 7.1 Metabolic Labeling with Amino Acids% Z7 u6 Q& C2 i& ?, j
3. Unit 7.2 Immunoprecipitation
4. Unit 7.3 Metabolic Labeling with Sulfate
5. Unit 7.4 Metabolic Labeling with Fatty Acids, j, |& i& R) O3 I% _6 @
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins3 P9 ~) I8 L3 b3 ]
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
10. Unit 7.9 Analysis of Oxidative Modification of Proteins1 ~. r: n5 Y, [8 J, ^9 ?
11. Unit 7.10 Radioiodination of Cellular Proteins1 O# C* s2 |' o- |
10. Chapter 8 Cell Cycle Analysis
1. Introduction
2. Unit 8.1 Overview of the Cell Cycle
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle9 P! i$ n/ }4 o, l1 l; u
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells0 L- Q0 C! l2 G
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry. t6 p0 d2 U$ X" _" E
11. Chapter 9 Cell Adhesion
1. Introduction
2. Unit 9.1 Cell-Substrate Adhesion Assays( z* q" E; i2 I! m# v
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion7 U+ W. q3 v3 |! T: H4 E
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions  P" {  v( Z" O& E% l7 i
12. Chapter 10 Extracellular Matrix
1. Introduction2 S$ d! C  f& c/ g% O- ?
2. Unit 10.1 Overview of Extracellular Matrix5 a- J" w' d. ]
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors. }0 V8 R* l# C' V: X$ ]
4. Unit 10.3 Preparation of Gelled Substrates
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9( o0 V0 C* b1 e1 \
Endodermal Cells
6. Unit 10.5 Purification of Fibronectin+ z( J3 n1 E" G0 ~' ?1 C
7. Unit 10.6 Purification of Vitronectin
8. Unit 10.7 Proteoglycan Isolation and Analysis& {0 R+ I2 p( h3 V6 A
9. Unit 10.8 Matrix Metalloproteinases
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts9 p2 c* }% [9 l8 M. s
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
12. Unit 10.11 Purification of SPARC/Osteonectin
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly1 H3 g- m% h9 G
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
Xenografts/ t4 \/ @0 K; Z
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning' Z. d% z. v# _" t& e" M  p
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties3 W7 p! u5 Q% g# j' B4 |
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D  x! U, H0 z3 l5 a% ?! G/ \) T
Cell Migration1 {5 w* B1 v7 U- y9 e6 z
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix" ]/ _5 @9 X; M- P- j( N
13. Chapter 11 In Vitro Reconstitution* F7 G; B6 m# e; ]5 J. c" N
1. Introduction
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems& T  _: A+ l# m1 L: N5 `% x- u/ i
3. Unit 11.2 In Vitro Translation
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes- z; O9 ?/ h4 x5 c0 _4 G
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
7. Unit 11.6 In Vitro Transcription1 D/ l" k6 {5 p9 E! y6 X
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells$ x7 S" D9 ~" {5 M4 [6 g  G: i
9. Unit 11.8 In Vitro Translation Using HeLa Extract
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts: P, b- `& O0 x% q
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
14. Chapter 12 Cell Motility8 U' l1 c5 `% K* |+ r3 z
1. Introduction, _! L5 ~( U, [" S9 s) I- \
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
3. Unit 12.2 Invasion Assays0 D1 Z' ~. z8 G8 w2 w9 G9 j% I
4. Unit 12.3 Cell Traction* S) \, ~  E! D
5. Unit 12.4 Cell Wound Assays5 _7 ?, R0 b9 v* U8 Q4 B9 m
6. Unit 12.5 Dictyostelium Cell Dynamics
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils9 y7 }; {8 t( [7 g6 X
8. Unit 12.7 Actin-Based Motility Assay
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
15. Chapter 13 Organelle Motility2 Z3 a2 C+ X! B9 |& o
1. Introduction
2. Unit 13.1 Microtubule/Organelle Motility Assays6 v) e2 @: r, f4 R. L# h
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin5 s% @/ D4 i/ L$ U4 F3 P: V6 O, |  w
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
5. Unit 13.4 Movement of Nuclei) F  E! f2 ?  `; i5 N) T4 w
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
16. Chapter 14 Signal Transduction: Protein Phosphorylation
1. Introduction5 W0 T' V5 L3 F8 F  x$ s2 G
2. Unit 14.1 Overview of Protein Phosphorylation, a% R, F. R: N9 t6 ^
3. Unit 14.2 Immunological Detection of Phosphorylation. u+ N2 B% \3 {+ j- l7 U% j
4. Unit 14.3 The Detection of MAPK Signaling& l* d* \: {- y' s
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation- B3 H/ R( |* k# }7 a( @$ ^
6. Unit 14.5 Phosphoamino Acid Analysis0 s3 _( J, G+ ~8 B( S( R
7. Unit 14.6 Determination of Akt/PKB Signaling
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events/ u: @3 @+ q2 Z
9. Unit 14.8 Rho GTPase Activation Assays
10. Unit 14.9 In Vitro GEF and GAP Assays+ l5 |& g7 s1 D2 E1 l1 {
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
Energy Transfer (FRET); P" x( @0 h1 I: E8 w, ]( P# S
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells) q6 `4 c" P1 |
17. Chapter 15 Protein Trafficking
1. Introduction2 G0 C- |8 d; @6 k
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking) Y! W5 M( u8 z
4. Unit 15.3 Endocytosis: Biochemical Analyses9 M2 {  |# s) Q
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation3 ?) U$ O& G$ k8 x7 Z7 [
9. Unit 15.8 Analysis of Protein Transport to Lysosomes: P+ z" h0 m1 g  h* j- n$ J5 @& u9 J: @
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells4 [! Q  h! Q* @
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and2 q: Q. s8 k% U# Q3 p7 G
EGF Receptor4 I" y- G/ q: L' a# Z; E% J
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers8 n$ k+ I8 g5 Y8 ^$ Y5 x- G8 k. O$ V
18. Chapter 16 Antibodies as Cell Biological Tools
1. Introduction3 Q" V/ ]* h# e4 F
2. Unit 16.1 Production of Monoclonal Antibodies
3. Unit 16.2 Production of Polyclonal Antisera# r) w- o/ k& x% c9 Q& y
4. Unit 16.3 Purification of Immunoglobulin G
5. Unit 16.4 Fragmentation of Immunoglobulin G
6. Unit 16.5 Antibody Conjugates for Cell Biology* o- \) ?4 `0 m  Z3 h. m
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides0 {- l  h; N, k! V
19. Chapter 17 Macromolecular Interactions in Cells
1. Introduction
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
Microscopy
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins- Z2 }- H5 u. h% g& H
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry) L: Z2 W0 N2 [1 i6 B
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors% ~" |# u) L) H' |0 p
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
Genomic Sequences In Vivo; j# ^! ~% L7 J: _6 A" `
9. Unit 17.8 Isothermal Titration Calorimetry  X" h, Q& r8 m8 r
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells) I% b4 t8 }+ Y( z* ], r3 V5 Q
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
Interactions (RFAP) in Live Bacterial Cells
20. Chapter 18 Cellular Aging and Death: t3 N! P2 Y" I9 e) r
1. Introduction) B$ ?4 B0 q" d" B3 Y& c' w; k
2. Unit 18.1 Current Concepts in Cell Death" F( m+ e% b& D8 y
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)8 H/ d; e) L. u& ]1 b
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death% v, `) T8 H- [4 X
7. Unit 18.6 Analysis of Telomeres and Telomerase0 D6 x, i& `: t, j
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of) q% |3 P$ l& E- \
Poly(ADP-Ribose) Polymerase$ h! N+ l  h* X) U3 L: ?% I
9. Unit 18.8 Flow Cytometry of Apoptosis  E) I1 u; V4 @/ a  Z
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
Assay6 X/ \( Z, a; |7 x1 x
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
21. Chapter 19 Whole Organism and Tissue Analysis$ F/ Q" g. X% E/ q
1. Introduction! h2 K. r( _  ~7 i% K
2. Unit 19.1 Overview of Metastasis Assays
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene5 O2 M0 Q( b. ]) P
Expression After High-Fidelity T7-Based RNA Amplification
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model* R5 C4 l% E; A% H% p+ w: S
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo/ f1 ^8 r! C7 |% K' i
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
9. Unit 19.8 Embryonic Organ Culture
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
12. Unit 19.11 Generation of Transgenic Mice
13. Unit 19.12 Overview: Generation of Gene Knockout Mice
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection* m0 }1 C5 ~( V: ?; I
22. Chapter 20 Expression and Introduction of Macromolecules into Cells! {* {9 y% n- b5 Z
1. Introduction2 G; h& t3 j: k; n
2. Unit 20.1 Direct Introduction of Molecules into Cells& O" C5 t2 v3 [  F
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
4. Unit 20.3 Calcium Phosphate Transfection4 Q8 }1 q' f% V' i# E
5. Unit 20.4 Transfection Using DEAE-Dextran- u0 U  o- {: V/ {
6. Unit 20.5 Transfection by Electroporation
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents. ]1 O/ N% G7 Y8 X/ z/ w/ b
8. Unit 20.7 Optimization of Transfection
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System$ s* w% g/ l) h2 s
23. Chapter 21 Fluorescent Protein Technology# K/ E5 Y4 A7 @5 R) w( h
1. Introduction" [3 P! L9 ^/ ~& Z7 u% S+ A( v
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells0 Y6 L) \9 a/ ]; ?- ], |1 k( p6 C
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence7 c. x* F/ |# r2 N% w
Complementation (BiFC) Analysis1 |1 ]  b9 e; L$ I+ X% Y
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology- f3 m6 P# h1 v! h3 o/ Z) W
6. Unit 21.5 The Fluorescent Protein Color Palette
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
24. Chapter 22 Cell Biology of Chromosomes and Nuclei
1. Introduction- G, p/ e. a. ^# \& o
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples" f/ c" @4 j$ k5 x) d4 |
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
6. Unit 22.5 Multi-Color FISH Techniques6 B: S. i3 T$ i4 k) e, J
7. Unit 22.6 Comparative Genomic Hybridization
8. Unit 22.7 Sister Chromatid Exchange
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery# `% C6 V  d: R. K8 H
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs: f1 [" j' c' z. ^: X6 t4 h, J% R  H
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly3 @7 k" Z6 R) P, p
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
14. Unit 22.13 Monitoring mRNA Export
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
Field Gel Electrophoresis  k: q8 E3 U0 d; W% e7 K
25. Chapter 23 Stem Cells
1. Introduction$ w8 ?$ @8 P9 K
2. Unit 23.1 Stem Cells: An Overview# K/ r( x$ h9 P1 T9 z( Y% @. `
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture  ]. U. Z* m8 u+ x/ g9 G* a6 J
and Differentiation as Embryoid Bodies1 k7 B- D, A1 {) @4 K3 z
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
Vessels
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into  j+ D9 \: r9 g% l& b
Adipocytes
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation. V+ Q" S- M' u; e5 [1 ~
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
8. Unit 23.7 Neural Differentiation of Human ES Cells5 g& a! t" n6 X! c
26. Chapter 24 Lipids
1. Introduction; o* a/ |. W5 x. k1 M. z
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking2 o% h5 c- b2 \, X) R2 N, g
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins! V6 G) L2 Z  `; C; V. L2 i
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film. I( T3 s5 _( \; V% {
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
27. Chapter 25 Nanotechnology
1. Introduction
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes, G- P* t) l9 r( D* i# `" r) g0 ?
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
28. Chapter 26 Viruses
1. Introduction' u" [# M: k1 V9 o# \
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
Cell Entry7 l4 X) b& U4 I; e9 a
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission, }# r1 o4 ^3 h! N" @* r7 Q6 E, v
29. Chapter 26 Lipids
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by. E% f6 g6 d9 Z# p, |6 v
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
30. Chapter 27 RNA-Based Methods in Cell Biology
1. Introduction
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible* ^6 f! Y0 W1 J6 M0 O3 S6 f: u
Rescue in Mammalian Cells4 ]' O, L7 R! x2 Y3 w+ t9 _
31. Appendix 1 Useful Information and Data
1. 1A Useful Measurements and Data
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research8 \* ], \& Q, P6 K- G# O5 E& y
3. 1C Identification of Motifs in Protein Sequences1 ?( e1 B! M1 O2 v: Z
4. 1D Safe Use of Radioisotopes, V: M0 w6 B5 h2 D
5. 1E Absorption and Emission Maxima for Common Fluorophores7 D& D( @+ c! \/ C+ `- C0 ?7 {0 T5 U
6. 1F Importing Biological Materials
7. 1G Centrifuges and Rotors  W- I1 D, H: d! [) C* i* K9 ^
8. 1H Internet Basics for Biologists
32. Appendix 2 Laboratory Stock Solutions and Equipment% ~8 `# _9 P$ {3 O1 \0 V; |+ W
1. 2A Common Stock Solutions, Buffers, and Media
2. 2B Medium Formulations
3. 2C Standard Laboratory Equipment
33. Appendix 3 Commonly Used Techniques. U$ W5 \3 P6 B! y
1. 3A Molecular Biology Techniques
2. 3B Spectrophotometric Determination of Protein Concentration
3. 3C Dialysis and Concentration of Protein Solutions( Y3 d6 I$ F0 Z+ n$ U3 T
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
5. 3E Silanizing Glassware/ j7 |/ X1 c  \3 E; F
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
7. 3G Micro RT-PCR
8. 3H The Colorimetric Detection and Quantitation of Total Protein
34. Appendix Suppliers* m  ]8 c* j8 |& f" v& H8 ^  P
1. Selected Suppliers of Reagents and Equipment
6 B: v2 w, S# W9 f

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