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- _# }. ?/ u* K, G! k' ]2 ]0 MCurrent Protocols in Cell Biology 2010年完整版 5483页
& c8 }4 M4 x: @. H9 f! _
) A& R# w/ {; b3 Z) u2 ?4 q% OOnline ISBN: 9780471143031
9 r8 ?8 ^# u% J% [2 i! O9 ?, tDOI: 10.1002/0471143030
( b4 w" g$ d0 S3 I6 f; h) {% m. _! Q; \! |5 f; H4 d7 O8 D
Table of Contents
3 v' @) e H4 \& @+ k: D: Q4 J1. Preface! S% J. e9 d$ O) y: A" D
2. Foreword2 r1 ^1 j9 q! w0 q/ E
3. Chapter 1 Cell Culture
5 i0 S, `( K& I+ c$ O* m5 n1. Introduction
2 |0 [( ^" x% Y; |9 A. Z8 W2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
& A3 E& `' A4 n% i1 f. O$ r3. Unit 1.2 Media for Culture of Mammalian Cells, i- e8 h8 b: s/ R( C
4. Unit 1.3 Aseptic Technique for Cell Culture
2 G, r; S/ A& J* v3 N, ~5. Unit 1.4 Sterilization and Filtration' ?- T" {1 M! {# [3 b" J, s# f n0 F
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
/ t( c6 ~( a4 b, x) w7. Unit 1.6 Media and Culture of Yeast n6 o/ n; s9 {# [: y
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging4 A) e* q* }- @- s. a4 h9 L5 C7 f M
4. Chapter 2 Preparation and Isolation of Cells
# P, z/ @7 {7 t* p1 q/ u' h; D1. Introduction
6 V8 i% r* I, U5 G9 H2. Unit 2.1 Establishment of Fibroblast Cultures
: t- U! Z1 y0 O0 y, t9 j3. Unit 2.2 Preparation and Culture of Human Lymphocytes
8 l2 ^9 }- V5 o% d4. Unit 2.3 Preparation of Endothelial Cells
& n7 ?( a/ i' g1 B( r1 ]5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation% ]: T) r1 ~& {; g# w" S) Y
6. Unit 2.5 Laser Capture Microdissection
7 P G9 m+ I- X; v7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
$ `. x+ B2 _# T0 A" v. p8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
7 }% ]4 n C5 S1 J1 n' e5 W5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
( g( F% M7 w; J) [: ]1. Introduction
* x7 b# ~9 F& y2. Introduction
' O+ d; d! q: {) o J* k9 b+ [3. Unit 3.1 Overview of Cell Fractionation
9 I6 t# P& s+ S5 q$ M4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains, m+ [5 H, b" e3 ?+ D
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation) M, C0 S2 C+ ^" v9 x
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
$ g# O, e+ t* a4 `8 _7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
2 \# a" ?6 \9 ?0 Z( DCentrifugation
5 U: _/ E5 d& W) x% A8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
1 v6 ~' ~; ^/ G1 vCentrifugation
# B# `; Q& [$ I; j6 W V% E9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae! \# k+ G% S" n2 i$ v2 a) b1 q) D
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
% O' s7 _) l M- y11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient: q& M2 v+ f7 j( }, @" n- V$ c
Centrifugation2 `: s2 S; P( ?" l' ^
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
) i! s! d2 Q7 A+ n- [! L13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes+ Z' b) m0 G+ u, z$ h. W2 F) d
14. Unit 3.12 Isolation of Synaptic Vesicles( Z' |) w8 z, {" I# i9 P' M
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
' O' {, o% A$ [5 \* U, U6 q: p, ]5 ^16. Unit 3.14 Isolation of Melanosomes
6 E* l: R9 V9 V$ _4 u' ]17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
4 t3 \+ n- c, [18. Unit 3.16 Isolation of Mast Cell Granules
& z* r. \( a7 A* _, Q5 z19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster. ?, e O& A* n1 o. O6 u
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas2 D; D0 p" B8 y6 t+ Q4 D8 {" o9 O
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
- |1 n1 U t* w2 t2 i, }/ M3 B22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
% C3 ^* f$ d- v( y# U& R23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
5 b( e& j- y0 a- d- t24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
! y0 n! g9 @; H @& ~Fluids
; f8 o; ^9 d& |; f25. Unit 3.23 Isolation of Intermediate Filaments
7 d( T4 r' r0 a0 ~# j/ N! J26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle4 h. I; U4 G) [+ B: ~
27. Unit 3.25 Isolation of Myelin# k, k( x8 Y. |. }8 J9 [
28. Unit 3.26 Isolation of Renal Brush Borders
# Y6 Y9 _3 d5 E3 z& V$ {( T29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane: k& F4 A: E" Z# z( y" g$ L% M
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
$ G3 ^0 C: V" E30. Unit 3.28 Isolation of Amyloplasts; k: G# l% Q) P0 H) Q/ |6 j8 g
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins7 z) [, {; R/ j4 V( C, B; g
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic$ D1 I( `6 J2 c
Centrifugation
- Z4 W9 s+ O) u0 ?% a33. Unit 3.31 Isolation of Neuromelanin Granules, f7 f/ Y: ^4 I* |/ b$ p, K( {
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
: x% |9 }1 p) B% ]/ F" e% p3 dDensity Gradient Centrifugation
2 j0 o2 A- Y9 j+ ~5 b3 y! I3 S35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
l" ?# H. \, q: p4 t36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
: j; }! [' g! Z Y' b8 B37. Unit 3.35 Isolation of Platelet Granules
) r1 T( K9 u- g# r* k38. Unit 3.36 Isolation of Nucleoli6 p7 B( ~; F- x9 P
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
6 ^/ A/ n) V l40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates7 l, ?6 O" q' t3 w' M( H
41. Unit 3.39 Isolation of Chromaffin Granules
5 ^9 _" j9 i m. |, g42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
% z3 O$ `$ X1 e6. Chapter 4 Microscopy. A$ x, A' x' Z, [. N& o
1. Introduction" ]' t) v2 x, \. |
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
" {% e8 T, B0 ^: s& c5 K, i3. Unit 4.2 Fluorescence Microscopy
1 c. h+ U3 J- Y: [( `8 G4. Unit 4.3 Immunofluorescence Staining; U6 j7 Z* v) y0 J8 }* M6 @
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria6 [' c, a, y1 W* a
6. Unit 4.5 Basic Confocal Microscopy
: k" f; M1 [. V# V$ L) l! i: _7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues7 \; J" w9 F& l, O- q% u1 R# W
8. Unit 4.7 Cryo-Immunogold Electron Microscopy7 R, K9 a4 A0 R# Y+ G. r5 x
9. Unit 4.8 Correlative Video Light/Electron Microscopy* e8 @& ]2 a+ I+ S# e. W& u& Z8 n
10. Unit 4.9 Polarization Microscopy' O& K1 Z4 s" v' P5 A* D3 @& J, T
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
% r; `' O' c, i# h12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
5 E+ g* x p9 ?1 k; ~1 R* g' N13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
: m: t3 Q4 f! p2 U" w" e' G: fEvents0 ^3 D! b% A2 v
14. Unit 4.13 Fluorescent Labeling of Yeast
% P' E# k2 ^3 n15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
' [, g$ x( @: A5 C9 J* i7 y+ }* Y8 Z, u16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy6 \5 j, G) h- A6 h8 @" w1 J
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules3 m. \/ X. m4 V. h4 W# s
in Endocytosis
& }5 f( l" v' T# E# b+ h6 ?18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
1 w" Y8 B& ]' H# D19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
. z' k7 o& u/ |& O8 U8 u; J20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images* ?5 {; |. c" K: B! h, q: Q' V
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
/ c% o( X7 W8 {! { ~22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes, ] k; z0 Q8 A' G9 j& _7 b% r% z
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics7 D5 {( G# j7 ?3 X L# r/ l
24. Unit 4.23 Interference Reflection Microscopy
6 [ ?# f; j+ ^- F) m# ^) ^6 N# [25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach5 `4 j7 ~; U$ S( o
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches" R1 w3 E! L! I# V; q/ q
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
3 w$ t2 V- q# S' H% D0 t' J) x7. Chapter 5 Characterization of Cellular Proteins
# }7 j- l5 Y; }3 y8 i+ P9 _1. Introduction
3 U1 F4 k, B; c# w; m; M8 V s2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
9 I" a& H4 g0 Q2 d. J& O3. Unit 5.2 Determining the Topology of an Integral Membrane Protein5 O: N7 ?( @' T9 N" |
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients! G# S# y7 S$ O) V7 W3 K: d
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
1 Y5 N* f) a2 T+ n+ J9 ]6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
* l$ g5 ]+ h3 O( u, \' A6 m* D7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
) z( R$ s$ q8 a4 R" bSpectrometry
, x6 ]- F, l# A$ ~2 M8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening3 L. M e2 d8 S7 L( s
Applications* M/ a4 `( b( z
8. Chapter 6 Electrophoresis and Immunoblotting9 y" i) F3 ]$ ?; I; J- ]1 x
1. Introduction' S; u7 {! A! l, D- U, s8 h
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins' ?6 I* {* h2 m- j5 D t5 Y
3. Unit 6.2 Immunoblotting and Immunodetection
3 p) A" s+ I" }4 K. G( q9 j4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
; w2 l( E* g% R- l" t$ k. Z5. Unit 6.4 Two-Dimensional Gel Electrophoresis
N0 [( Z z1 v% F6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
F/ T0 r; s. m+ B" M$ M' e, v" z7. Unit 6.6 Staining Proteins in Gels) u/ `( H+ D4 U" f- j
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins$ U3 j3 n2 R9 Y' ?2 V2 J$ n; S1 o
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots- }, b( t5 O6 H1 R' \. t0 G
10. Unit 6.9 Digital Electrophoresis Analysis, B) Z! U$ b& I# C4 g
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
8 d7 n5 w. o( `; E& d( b12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of S6 ?- R* ]- Q' d
Single Cell Gel Electrophoresis (Comet Assay)/ s* F" j7 {5 E
9. Chapter 7 Protein Labeling and Immunoprecipitation
# e$ k! O; ?4 W. X1. Introduction, h0 o. R( I7 F
2. Unit 7.1 Metabolic Labeling with Amino Acids
5 n; W! P6 J: ?# i- r1 C7 s3. Unit 7.2 Immunoprecipitation
6 u* r5 y+ ^/ p9 g. x4. Unit 7.3 Metabolic Labeling with Sulfate- X+ h' t" g: k+ t8 e6 S0 n
5. Unit 7.4 Metabolic Labeling with Fatty Acids5 `, D/ g7 x1 f6 S8 M8 I
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
5 j5 T7 I; K! V# m/ P7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins! R+ }# r) l+ @
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
* Z! ~' K2 t/ w; g& ~. D9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars! B5 [9 D0 k# G; x8 `% H9 \+ D
10. Unit 7.9 Analysis of Oxidative Modification of Proteins$ O. U$ p2 B- u3 R' `& H
11. Unit 7.10 Radioiodination of Cellular Proteins
# ^4 q0 h* K) U% a4 B) b10. Chapter 8 Cell Cycle Analysis5 K( a, V! j. X( w& D8 E$ w
1. Introduction' y3 i" i/ R8 V, J! t' x
2. Unit 8.1 Overview of the Cell Cycle& j3 z. n7 ~' Y3 I2 w, u( H
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
+ U4 T- r4 z# m6 }' P& V4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
+ f* c. U9 f7 l5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry; r' i' ~! H! I( \6 u. |
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
, ~2 s3 a8 X/ }4 r7 l. t# V7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
/ ?. |9 C6 m8 n11. Chapter 9 Cell Adhesion
( O c: l/ ]+ q* @0 J6 k1 H1. Introduction
& H" \7 B2 ]3 m! K2 Y- ^5 i( b2. Unit 9.1 Cell-Substrate Adhesion Assays X) o9 V/ ?2 T! [7 G6 @- A
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
& K( R! c% i: N& B4 B H9 t4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion. E+ N/ b& y( Q. V0 x% J' F2 N
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
1 B' D% g8 {: Z. b5 f/ P# D+ N6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules P: b$ F) X/ K" M& X6 w& T. o
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
$ z1 V: L0 M$ X/ I12. Chapter 10 Extracellular Matrix
( [ p% I8 F# W5 q. E6 a1. Introduction
& Y8 c* u; r% g. ^2. Unit 10.1 Overview of Extracellular Matrix
, A' l' g# w F! q8 ^, Q( h; ~3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors F$ ]- P. d" L2 F
4. Unit 10.3 Preparation of Gelled Substrates1 d5 G2 o+ M# v( R- D
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
$ \! W; L: s9 m1 s$ h" J, OEndodermal Cells8 {: `& ] n: _2 [
6. Unit 10.5 Purification of Fibronectin
$ G7 ~& Y8 k- k, m1 c7. Unit 10.6 Purification of Vitronectin$ I# @+ i5 d# r. r5 w$ Z
8. Unit 10.7 Proteoglycan Isolation and Analysis
$ u' b H3 W$ D' f% v, u5 _( D5 I9. Unit 10.8 Matrix Metalloproteinases
+ `3 b. C6 p9 W9 w* b10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts$ e) L( ]- z$ o! l0 i
11. Unit 10.10 Purification and Analysis of Thrombospondin-1& i* i* c6 I) m" U+ [8 Z
12. Unit 10.11 Purification of SPARC/Osteonectin" B4 u0 J: h5 E& P
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
" _$ B1 Y* u2 D* K8 n+ N14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly) Z: q/ i6 d6 {& U
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor8 c$ H, |6 I& d; x' p
Xenografts7 d' y! t0 r; A9 B
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
2 T$ M, k$ f: C F; z. I17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties8 ]2 g% `* l/ q. X L% n! a0 x
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
: N& A. e" X, g2 j* k; gCell Migration
8 Y7 R/ ]; i" [1 e1 a19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
( m4 w$ T- u& \7 P) ]# [7 U13. Chapter 11 In Vitro Reconstitution' o8 _& U; r7 \$ j' P( b. x
1. Introduction! [& e0 v; U+ C
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
1 r4 L I+ w( L+ M3. Unit 11.2 In Vitro Translation5 N3 U3 z: r5 r4 X
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells+ ?% Y+ {! I \5 B% O
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
& z, O3 H: ?0 ]8 K9 R5 q* k2 `6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
3 N" E* L7 T$ V7. Unit 11.6 In Vitro Transcription$ [ {3 M g, B% Y
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells) i0 x* R4 T2 n" L$ C- ~
9. Unit 11.8 In Vitro Translation Using HeLa Extract
4 f( a- q- z! K: \% j( }10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
" p" y5 ^: R! x l11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 a; u! j8 V3 _% Z+ c
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts4 ^9 X& a( Q- g( V, V, R# B
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
3 j9 |" t& M6 {- ]& e. \14. Unit 11.13 Mitotic Spindle Assembly In Vitro
' X* C- I# n/ s15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
6 T, j F& f9 `" W16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
7 S0 e) V! U9 n; M/ [9 t1 r17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import2 u' a" y* ]& G7 C8 b
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
3 w6 p) a/ N$ d" N; b19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells+ [7 e) X, l. }1 Q2 W& }
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import# u$ X0 O5 ^# o4 T
14. Chapter 12 Cell Motility2 _- d" W. B% u
1. Introduction( a& o/ `, a4 V
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells! y1 q/ Z% G8 c% S
3. Unit 12.2 Invasion Assays
9 |$ z$ e9 J) S- D' y4. Unit 12.3 Cell Traction* F- _- G- ? b j$ g9 W
5. Unit 12.4 Cell Wound Assays) C9 `5 p H6 z3 }% c
6. Unit 12.5 Dictyostelium Cell Dynamics" N+ q3 z% i$ d% f0 A7 A4 q
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils& B8 Y1 L( R3 v1 Y
8. Unit 12.7 Actin-Based Motility Assay
5 g: A. [' d- V# C9 }+ X9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP! R0 i" U, X( s2 g9 \( A* ?
15. Chapter 13 Organelle Motility5 H* ?% R5 Z% n/ ]! P
1. Introduction
( [0 w! L# p k; J9 r6 C2. Unit 13.1 Microtubule/Organelle Motility Assays
4 F$ I* W% m1 P. s3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
& L8 M6 k. m1 H5 i! L- V1 J4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP t1 t4 n& D! u N- I. O$ A- j
5. Unit 13.4 Movement of Nuclei; W4 V7 V. \9 {: ?; g# o- b: q
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching% @9 q1 |0 Y, l% I
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly3 W1 l2 R$ Z- n# p+ P3 K
16. Chapter 14 Signal Transduction: Protein Phosphorylation
" t. {. F# t9 i* P( k1. Introduction+ ^6 u( H) Q4 ~% k- ]
2. Unit 14.1 Overview of Protein Phosphorylation0 T0 U3 M9 D" c
3. Unit 14.2 Immunological Detection of Phosphorylation J1 Y# |. ?8 K/ ~
4. Unit 14.3 The Detection of MAPK Signaling
, g" k3 K' n1 b) w5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation) h" h3 ~& `# @) k! f+ j: ]# o
6. Unit 14.5 Phosphoamino Acid Analysis& `' u* ^5 y6 C% U3 |
7. Unit 14.6 Determination of Akt/PKB Signaling
6 V8 i) x3 v' R) I* g8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events9 j/ d2 |* x* D! Q. k/ d0 Y8 h
9. Unit 14.8 Rho GTPase Activation Assays5 X) l. }( M1 u2 F
10. Unit 14.9 In Vitro GEF and GAP Assays5 n0 D3 z7 f& F2 X9 x( O
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
4 `" f$ O1 n/ T/ S* _# cEnergy Transfer (FRET)8 |+ H |) n/ C) {* f4 B2 c( b
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells# k# p1 w4 e/ `: T7 [1 s
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells: x7 l/ p/ D% ]- r3 C
17. Chapter 15 Protein Trafficking8 p; n7 A; L4 p7 S5 D: g2 J
1. Introduction, y# h" j8 D8 `; W* O: [
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
7 i/ F+ C5 P* U) C6 t3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking" S9 H7 [6 k9 c
4. Unit 15.3 Endocytosis: Biochemical Analyses
, \' u( w% y# c4 v7 h1 F5. Unit 15.4 Determining Protein Transport to the Plasma Membrane9 j9 M, j8 `/ w% z* L1 E" K
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
. c, v2 S6 z2 o8 F2 v7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum: I$ \/ `; a. M" P
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation! ^* y" e4 K( C; c( V: k
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
) ^" P3 W3 M9 d10. Unit 15.9 Studies of the Ubiquitin Proteasome System% ?0 i6 f- W `$ ^/ q
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
2 w. V- O" j! R/ } ~12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules; Q- w3 v! d; J i
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
) D& t' q" B* P: Y5 w- n14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
. m4 @" S* f- S* E: _15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and/ n: A. Y( b- I( S
EGF Receptor/ d' d* G+ @ _4 ?# K
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
3 c; A* U D- m* p0 I18. Chapter 16 Antibodies as Cell Biological Tools/ p r6 M |) B i
1. Introduction b# g9 Y6 d5 N5 R2 R
2. Unit 16.1 Production of Monoclonal Antibodies! u; z. p4 q+ I z& l; _
3. Unit 16.2 Production of Polyclonal Antisera
& P& P% S( H3 K9 l- ?9 N4. Unit 16.3 Purification of Immunoglobulin G
+ d) D' Q+ N! G5 w2 W- S. l5 l5. Unit 16.4 Fragmentation of Immunoglobulin G9 M: \0 ~4 |8 H: t2 S
6. Unit 16.5 Antibody Conjugates for Cell Biology
' e- ~- S" ~) [, Q. f1 L) A7 { W7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
- w/ I2 n) Q8 P. ?9 F6 ]19. Chapter 17 Macromolecular Interactions in Cells1 h2 V7 U0 X5 ^- Z& l4 b
1. Introduction! c( ]: r0 q. |% N. F/ F7 Y/ r
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET); ~- u' n& |' D5 p$ a; }; N
Microscopy$ n3 K3 \9 z/ S6 Y& Y# z
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis% {& @4 e7 q# M# V- {
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
' }, c: X, w7 Q5 i5 {3 u2 Y5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
! x& W, \" z& C3 a; o5 C6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry1 S0 _; J4 b p1 E5 G; f
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors4 F4 T/ u: B. }" b: {" \ j* o7 P7 |9 E
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
5 u& v9 P4 V8 R: i5 M C. o5 PGenomic Sequences In Vivo
5 } G' X( H( e4 e9 Y2 l9. Unit 17.8 Isothermal Titration Calorimetry+ j5 r: @3 w5 F# q2 k; K% B
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
\$ ?' S3 e: o% |+ d9 \7 F( y11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
0 j' a. Z( h1 g# b' F/ J7 x1 D7 W12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein, p; T3 }& m) a6 ~: D9 ~
Interactions (RFAP) in Live Bacterial Cells
7 B( W( o+ Q4 R5 R6 S$ _6 b20. Chapter 18 Cellular Aging and Death
* ?0 T9 B$ A. V) u& [* z- R1. Introduction- {! P2 c. A% g! X1 |" t- _ T
2. Unit 18.1 Current Concepts in Cell Death
. s( _2 r1 J7 O3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
6 q- _& W1 [- V) I: n4 D4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
) h2 f8 v- Z# k* D5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH), |, e! B- Y6 T+ a* r$ G
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
m& B6 o: y. o7. Unit 18.6 Analysis of Telomeres and Telomerase
- P0 Y- G5 W0 B% X. c0 {, K, n4 d }" O8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of ~* ~% ~9 I* S4 a- @8 J( ]
Poly(ADP-Ribose) Polymerase
$ _, {" t; u d, H; A, {9. Unit 18.8 Flow Cytometry of Apoptosis/ g/ w+ C4 x2 i C2 y. V
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
# `5 a+ K$ c# cAssay
: {7 A0 { c! d11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
# I" U6 s" I6 M21. Chapter 19 Whole Organism and Tissue Analysis
4 P8 w/ ~1 G) [2 T1. Introduction5 l4 `6 l% L4 E
2. Unit 19.1 Overview of Metastasis Assays! ~7 M" b& i/ c; ?& a$ p4 A; p
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
! j; x0 O$ y* h) l( h8 j2 l: }4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene$ V7 g2 @0 z/ Z' O2 F9 X T
Expression After High-Fidelity T7-Based RNA Amplification
( z! J) P* j$ M* Y) k5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA1 C& P* g: i; `+ s, U- J1 g
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model6 C) |' v/ X% z. A
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
) @" c5 |" ?. y) [* d8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
2 Z. J; r# Y/ s9. Unit 19.8 Embryonic Organ Culture$ z4 Y/ ?8 o0 v+ R' j# a. r; @
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
6 `% k% N9 K4 S+ B5 W11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
7 o1 y" C- Q! }( E1 e3 q' W5 n12. Unit 19.11 Generation of Transgenic Mice0 S5 O( v! m: b2 c
13. Unit 19.12 Overview: Generation of Gene Knockout Mice' a# [/ T- D" C# L- j, {- l! u$ K5 S T
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
* D9 ~# c. G4 z4 w; Q" @15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
: X& W" u. i; W- Z- s. V22. Chapter 20 Expression and Introduction of Macromolecules into Cells' k+ Y" K6 O7 I) h
1. Introduction
8 J% s, l% o4 i; L; O% h) B) Z# Y2. Unit 20.1 Direct Introduction of Molecules into Cells
# n( H/ a4 P" o: Y, f1 g2 d3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System/ [8 H0 r1 h* b x
4. Unit 20.3 Calcium Phosphate Transfection
( N6 `2 ]5 N5 ?9 k! ^" n# |; ?5. Unit 20.4 Transfection Using DEAE-Dextran6 k- z3 Q% i7 T) _2 i' P# |% m
6. Unit 20.5 Transfection by Electroporation4 y3 l$ B9 b+ C) k! }
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents9 g. }3 ? \" X! o) \6 t
8. Unit 20.7 Optimization of Transfection8 O* n: ^; J% Q4 u" h
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
7 n0 A; u# r6 k$ R23. Chapter 21 Fluorescent Protein Technology! B. q4 t/ |- v) u
1. Introduction
8 \* ?5 s' Q! Y, k) k& U2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells9 s9 [# M! H0 f! C0 F
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
& P8 ~+ a; ~! J# N+ Z. Q4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
6 e( Q) O6 j* E! UComplementation (BiFC) Analysis
8 K2 j& i! a" p0 x: H; w4 ^5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology2 B/ p5 e x% `! S4 k
6. Unit 21.5 The Fluorescent Protein Color Palette2 I l1 e2 W8 J! Y/ P
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
- X5 h6 u- M/ v( j7 u24. Chapter 22 Cell Biology of Chromosomes and Nuclei
- X$ F3 W- s+ t# f* n1. Introduction
( b, X/ u( U* j2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis- }8 m3 g$ Q2 B, t8 }3 }" G
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples0 E! C+ Y* o1 X. K! N! i
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
5 g, v+ E, [# v' E) d4 e* I) p; h5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
/ |+ M# [7 [4 e6. Unit 22.5 Multi-Color FISH Techniques
7 H; @% N. V# o: N7. Unit 22.6 Comparative Genomic Hybridization
: ]- M: ^! z5 m% c, u. `' q O8. Unit 22.7 Sister Chromatid Exchange; E N. E0 L$ z$ t$ v
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
6 E6 K) `& b+ G2 G0 Y10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes, r) `6 [8 }) D% O2 @& \; \
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
+ s$ ]' X3 u& {" p4 [; \7 E12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly6 J6 x. ]. L7 h. K9 g I
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells/ P3 m/ i2 K0 k- I
14. Unit 22.13 Monitoring mRNA Export
7 O7 {) n! [& \! V4 R: B, t15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
" a9 O" \* j, I k2 vField Gel Electrophoresis
1 w! S1 K/ Q- @9 `9 J, `25. Chapter 23 Stem Cells/ o; m5 e9 ~( ^8 S5 y% Q- z
1. Introduction
4 `/ R* J% A) q1 h' @5 d2. Unit 23.1 Stem Cells: An Overview5 p! Y0 Q6 B9 w+ v1 H
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture# F) F0 w* H$ q2 |9 R4 [
and Differentiation as Embryoid Bodies: p4 ], [% U" L d2 m
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood* {1 K. l0 R/ ] @& c
Vessels
1 O0 f$ j& j) N1 F; @+ C5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
. P5 M6 ~: N( V8 w3 lAdipocytes
2 J- z) [( `* d8 ]8 @0 z. H4 |$ W6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
+ g' L1 M/ n% q8 [4 d' p& m" ?( |7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells( L; L6 ?( @6 | I0 m! `
8. Unit 23.7 Neural Differentiation of Human ES Cells: M+ Q+ ^) J; k5 u1 Q
26. Chapter 24 Lipids9 j' F2 _3 l& z5 }( I& ]6 O
1. Introduction2 u: Y+ }$ ~. S8 i6 f, }- h
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
0 B% h" A3 A- g7 s3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins' V+ G6 l% N* T$ H2 V
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film7 p# `* \7 s* l* Y8 Y; X$ z/ T
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains) H% _# M$ J- E! B& @3 S8 c9 i
27. Chapter 25 Nanotechnology
5 B, B- \. _% \1. Introduction& G$ N/ Y( |* F$ b8 g0 c6 R
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
* r: g5 m* [) ?: a3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering# ~! d5 t( v4 y, F$ w8 K; Y+ ?9 J
28. Chapter 26 Viruses9 z" D3 e& i Z9 J8 {
1. Introduction' _4 `' H' L, _8 }
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
: J5 x* S$ p1 \/ T- {- V; c3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
! j& g7 ~) V% n% f( p5 | c( `Cell Entry P2 L6 S5 \; z! M4 N8 }/ h# _
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection, \( d+ Y) B4 ~
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
3 r7 ?( {! Q, F' z6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
' |9 z5 u, ?# [6 P29. Chapter 26 Lipids4 O1 y- `5 \7 f/ ]9 ?5 c
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
. }( S- J0 c, Q" ?8 u, {4 [$ W" hReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication# x9 H4 b, `! e: V) |& u. o
30. Chapter 27 RNA-Based Methods in Cell Biology8 X, b8 P/ ~% b4 m
1. Introduction8 w' t, @, a# a* x9 W2 M s
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs h) {- z+ l6 X
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
% z* l- S% Z9 c' P6 hRescue in Mammalian Cells+ v" ]. M* L4 c
31. Appendix 1 Useful Information and Data
: Q+ G: f9 Y6 q! e) Z1 L1. 1A Useful Measurements and Data
% j# y+ n7 N" A6 Z) D; D9 u2. 1B Compendium of Drugs Commonly Used in Cell Biology Research* z; L: L+ d0 m& l+ v# N6 X' f3 Y
3. 1C Identification of Motifs in Protein Sequences
) y5 u! R' [3 P4. 1D Safe Use of Radioisotopes! V( G" M: ^4 M
5. 1E Absorption and Emission Maxima for Common Fluorophores6 h, _ n9 _- n
6. 1F Importing Biological Materials, \; ` Q- H8 g b- h! E
7. 1G Centrifuges and Rotors
# |0 Z, z0 f% o! b9 e8. 1H Internet Basics for Biologists
6 X' i/ ?2 v! W: Q3 V6 C9 `: K32. Appendix 2 Laboratory Stock Solutions and Equipment2 g8 E7 I4 H8 A: J+ F; @* ?! q+ W& b
1. 2A Common Stock Solutions, Buffers, and Media
" L; K9 n6 f, o" s9 o2. 2B Medium Formulations# H z, x$ _9 y' F! b& W
3. 2C Standard Laboratory Equipment4 o3 L8 n0 I# J& N
33. Appendix 3 Commonly Used Techniques* W* S( \- j/ c/ D
1. 3A Molecular Biology Techniques
, s2 V* t5 h& k5 L- g2 W3 R' A2. 3B Spectrophotometric Determination of Protein Concentration
& t( W, W9 F5 i( K3. 3C Dialysis and Concentration of Protein Solutions, n! s0 m) I F, c6 M: \" r J, F
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
$ N/ d Y# _( N/ Y6 i6 O' x5. 3E Silanizing Glassware/ D1 h) Q# B4 J* M. J" ?
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization( L8 M1 w7 ~- c% ~; l/ T
7. 3G Micro RT-PCR
- I, F' K5 m6 R& Q5 d, W( i* K0 p8. 3H The Colorimetric Detection and Quantitation of Total Protein
3 M* B$ g1 T% i; V34. Appendix Suppliers" Y# K% Z$ i' T5 b% u
1. Selected Suppliers of Reagents and Equipment# L3 k4 z' j. \; K: o3 N
7 P, X# T0 q w5 |3 F |
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发表于 2011-3-8 15:53
