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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 0 j* ~3 O, P2 u- w2 p
7 E! Z* p3 t: \9 \, c3 c* G) dCurrent Protocols in Cell Biology 2010年完整版 5483页
' S4 [& q8 O! {8 Y4 t) E0 b; K3 g- ]! ~- d0 k6 H8 [% Y5 V
Online ISBN: 9780471143031% Z3 x8 k* }+ }/ z
DOI: 10.1002/0471143030$ J& N4 m7 L: ] _( G/ A
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Table of Contents# n) S! J7 |: H2 M7 m- C. \) F
1. Preface3 X% W8 k7 C H& t
2. Foreword
* Q$ P0 R# ]- n# r) B6 V$ A: s; X4 E3. Chapter 1 Cell Culture
! O' L$ {; E$ L2 x8 I/ i# a/ V1. Introduction' n, F3 |, F' w0 Z8 D: `7 C
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
- k2 d. z$ ]6 Q( L3. Unit 1.2 Media for Culture of Mammalian Cells
# r/ a5 L+ ]8 u! {2 ]4 c4. Unit 1.3 Aseptic Technique for Cell Culture
0 l( K6 D; c0 }5. Unit 1.4 Sterilization and Filtration3 O' W- B# w$ I" ^
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures8 L r# [! _; ~8 o; u
7. Unit 1.6 Media and Culture of Yeast4 W& y0 i4 c/ m- O/ P2 n# C
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
5 C5 N: _2 \6 }, u- ~1 @1 B+ x4. Chapter 2 Preparation and Isolation of Cells
0 s9 ~% P t' C8 ^4 Z k1. Introduction
7 ~' h8 F& e# n3 K3 w+ B2. Unit 2.1 Establishment of Fibroblast Cultures
6 Y) m# \5 Y; y( w" G, U3. Unit 2.2 Preparation and Culture of Human Lymphocytes
( A( @* `. R9 w% O5 O0 @4. Unit 2.3 Preparation of Endothelial Cells
' N( X; f# @. Q' S8 D$ S7 j5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
% `7 R1 ^" {( N2 Y. S+ M6. Unit 2.5 Laser Capture Microdissection
+ b0 j% j' K0 R) O- e, r* W+ B2 T7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures. Z8 N5 U5 Z9 U: J
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells. E2 m" \3 g: N+ I) W" r
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles8 d. @: V2 u' m
1. Introduction
# o9 V D5 i9 ^; _1 n; |2. Introduction& a0 s8 v/ S4 U' t& }" c- d3 R) G
3. Unit 3.1 Overview of Cell Fractionation$ _# ~( E( d4 ^$ ^
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
6 L' u9 N' ~( F# j6 I" p* X! c3 R5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
/ k9 t$ O2 l) S5 b6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
: B; ]1 i) }: p9 q( j7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient- E: D, n+ _3 B1 `4 o9 t) {8 D
Centrifugation F) c( Z3 E% N# X4 I
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient2 s9 \5 p( ?, `( R: p& h8 ]
Centrifugation2 b4 q3 |9 E! P
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
k4 Y/ B& ~2 e: V( x10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
& O ~$ T' f! u& s: ]11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient( ?. X( L$ ^# K7 Y, h: n' X
Centrifugation/ @$ Q9 k$ a3 N0 E% x
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues9 B6 _5 H5 r" z3 O4 \
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes3 n$ h8 H4 c) I% n6 o0 }
14. Unit 3.12 Isolation of Synaptic Vesicles
+ U. f n' h" B1 U15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
; J% l* W# B# j! o! j, }- _16. Unit 3.14 Isolation of Melanosomes& x( r$ S; W( s& Q y! V
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation. h7 b6 s" ^9 a( }5 v1 a
18. Unit 3.16 Isolation of Mast Cell Granules. p0 k1 e1 y' G3 N
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster7 p% u8 d3 B6 A( Q9 R d, [, ~
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
~7 e5 T+ a9 F$ k, {% E21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
) @8 t# ^7 O' `22. Unit 3.20 Isolation of GLUT4 Storage Vesicles7 `+ C! y t4 M8 d
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
& q+ e2 K7 r4 K& a2 n2 R' D% n24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological) Y6 {; P) @0 P5 I$ I, n# r) f
Fluids$ }9 k8 c- y0 w& Y1 y
25. Unit 3.23 Isolation of Intermediate Filaments
5 ^+ c$ m0 y* ?, A2 S+ F26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
h# X- ?& r$ P$ [27. Unit 3.25 Isolation of Myelin
0 Z8 @% B% ~( G0 m. j- o9 V9 I( r0 k1 ]28. Unit 3.26 Isolation of Renal Brush Borders8 |& Z# H$ b6 `& K7 E( X
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
2 P4 g3 z1 C: w7 L, O3 i0 E: GFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts, Q6 f1 u$ X1 ^( |
30. Unit 3.28 Isolation of Amyloplasts
4 W' D: z7 L3 O+ [7 e( `9 k& R5 c31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins. ?0 N$ x; t' ]) {6 u" B6 }0 N
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
7 M: e( D* r! Y2 Q+ G8 lCentrifugation
8 S8 W5 L- t* W1 c+ {! y& r- d33. Unit 3.31 Isolation of Neuromelanin Granules
' E: G7 ^: b7 E) Z. _8 j4 [34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
, M% [. S9 E; ^# EDensity Gradient Centrifugation
# N' U9 D9 }, O, V; f" E* k9 v1 v35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
" Q% W& r/ Y0 B2 I+ D36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
% x) ^4 g1 D. g( X& p$ |37. Unit 3.35 Isolation of Platelet Granules
8 T; [1 L; F9 U' ]/ s38. Unit 3.36 Isolation of Nucleoli1 r1 k; e }+ O+ ?! F/ q7 s
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
# E3 N. I2 ^: |2 p& C40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
+ q) E d5 U" O. p& n41. Unit 3.39 Isolation of Chromaffin Granules3 b( \: \. I6 T- r( {5 C
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
; {' s' n# X( v$ S; i0 z$ {6. Chapter 4 Microscopy4 k& M+ S; z7 D1 w- _' Q1 t
1. Introduction
4 R# V# A# m- F4 C0 @+ V2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
6 }( _4 A; l3 b+ j& h3. Unit 4.2 Fluorescence Microscopy5 |/ q$ E" W7 U6 z' V+ `0 ?
4. Unit 4.3 Immunofluorescence Staining
4 a7 c1 c$ p$ @5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria. B9 j1 d$ I$ j" Q
6. Unit 4.5 Basic Confocal Microscopy
/ \, x0 W ~: M* D2 ?2 {5 \! ]7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
% u1 p- O1 J; E( C' J6 a8. Unit 4.7 Cryo-Immunogold Electron Microscopy' c2 U: y7 V. j% L
9. Unit 4.8 Correlative Video Light/Electron Microscopy
7 m& e0 S( [% a5 U! g10. Unit 4.9 Polarization Microscopy
- D- ^$ S# S2 q1 w1 q2 N! r11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
. W N/ t2 c5 y; M: w12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
4 F/ n h2 }' ?( M13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface2 w k8 q3 y, t0 g
Events
/ t! A8 m# u- R% J% v14. Unit 4.13 Fluorescent Labeling of Yeast8 ]0 u# F, A$ b2 Q" ~: F: k. H
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
: }& z* m! c* j& J+ O16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
/ S. b- Y# ^ d# H) v6 \- N0 A, V17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
2 m# \. I# o- m" r4 N. z3 Xin Endocytosis
. ~( g% i; E+ O. h- }$ I$ \18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
* F/ F! g' F# V" T8 k19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
. M: a7 W7 T+ W9 _20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
; f: o* [( G. F( H4 G21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions+ J4 e; G. z5 d6 @/ r& ?
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes& X# Z" v( Q. j
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics- {) v; R) k. |* W% E( x7 m
24. Unit 4.23 Interference Reflection Microscopy
* Q8 x2 ]9 r- n" i# \25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach6 l0 v0 s$ ?; o7 v; p+ F o) Y
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches& G4 B- h: ~. M* b. [7 ?) _, Y1 o
27. Unit 4A Organelle Atlas: Appendix to Chapter 4) m. R# @; X. R3 u
7. Chapter 5 Characterization of Cellular Proteins% F% ~! k* v, o( ?. V
1. Introduction
$ R+ M- L: T2 W2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
4 N) G& E M5 [2 ^% G: H' S3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
8 `) K; _& Q E+ m7 ~, v4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients0 q; B! @" m6 `- q: r
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
: e; B7 D/ O: C5 c" f$ I6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration): I2 Z3 j/ R9 O2 |6 \" [
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
' P5 x7 q/ |7 w% vSpectrometry
& k7 ^' H5 b/ s7 G8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
' Z' j0 M& {2 B7 A) p6 iApplications+ T, V. a; _- O
8. Chapter 6 Electrophoresis and Immunoblotting
: C; G7 B: n- p8 k1. Introduction: o( T8 F) @/ E9 Z( ?, s' C1 Z
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
- Y$ q4 N9 |* u, b3. Unit 6.2 Immunoblotting and Immunodetection
1 |% b% h$ P9 J6 }4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
2 v5 p. o9 f- C6 z" j$ _5. Unit 6.4 Two-Dimensional Gel Electrophoresis
, s# n/ l7 ~6 `* A6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions; l. m: u4 D( L% A2 K4 K
7. Unit 6.6 Staining Proteins in Gels6 r+ L& J7 u& `2 |! D8 d( T
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins! d$ B) t. ?0 Q1 Q/ V
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots- h% D6 t' _9 x( c
10. Unit 6.9 Digital Electrophoresis Analysis
) B ~8 r& q7 Y" A! V' Q5 K$ ^( \11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis: a0 m* U9 Q$ R3 [+ ]
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
t/ }9 H6 i3 s0 S2 qSingle Cell Gel Electrophoresis (Comet Assay)! a, @% c1 j9 P2 {9 d
9. Chapter 7 Protein Labeling and Immunoprecipitation
+ ^4 ~! q( n& a5 s6 E: w& G1 B" F1. Introduction
) m0 A' h* b8 T" O2. Unit 7.1 Metabolic Labeling with Amino Acids
8 a8 g) |* S5 U' q: d3. Unit 7.2 Immunoprecipitation
+ C% K9 c& t5 M4. Unit 7.3 Metabolic Labeling with Sulfate
Z$ j8 X- h# { ~" g5. Unit 7.4 Metabolic Labeling with Fatty Acids
3 V) i5 w! W7 D1 N; l& n; N6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups& |* \) ^# T, c$ C* j# F: R2 V
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins$ ]* y, O5 ~( `
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins/ c" E" V+ R9 `
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars9 j T/ z6 g/ W
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
{' y0 y0 ` @5 _6 M" A+ K7 L- r11. Unit 7.10 Radioiodination of Cellular Proteins' I+ }' A1 @. i& C8 t
10. Chapter 8 Cell Cycle Analysis/ x+ w" O" ]- f: r3 V: I) j
1. Introduction4 d( [& y; }. q9 D
2. Unit 8.1 Overview of the Cell Cycle
4 k6 ^' h* P. U1 q8 A1 A$ Q3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle+ E! ]4 l( C, B/ J4 [0 B
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
" m9 \& v0 h1 Q, J4 @7 c5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
$ [: z6 K: ?/ b) Z0 m& K- l* t6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
4 q9 ?9 [! S5 Z) d3 w, M7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
' X4 s6 H1 A3 Q1 d: h2 f11. Chapter 9 Cell Adhesion' Z9 W9 u* R7 `( j; h
1. Introduction
1 I6 q' T$ y& { K8 H/ a2. Unit 9.1 Cell-Substrate Adhesion Assays
2 U6 }6 |+ O8 j o" Y3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force6 h' Q3 v% }, r6 Q+ X5 N% y. R
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
# Z5 h' r7 k3 Z0 z9 u4 A5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
+ c6 f( n2 G( B w/ A% w5 m6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules) F: `* U" y7 ~4 v+ m9 Y) z$ V
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
( s& y4 t/ {+ G/ c7 Q" X12. Chapter 10 Extracellular Matrix
/ \- m7 W* J* t, U8 F/ |1. Introduction$ Q& U; V, X) E7 A9 X* t6 }, @
2. Unit 10.1 Overview of Extracellular Matrix8 |$ f9 _; P! g, h8 y3 ]
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors; X+ ~& H: p# C
4. Unit 10.3 Preparation of Gelled Substrates6 k) C5 W5 c; P2 E
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9: z) s5 t& C2 m6 F. ]8 u% c2 a x
Endodermal Cells- J: Y1 F7 h* ]* B
6. Unit 10.5 Purification of Fibronectin
$ e+ @$ ]6 |8 Z7 h5 b' V7. Unit 10.6 Purification of Vitronectin; Q( L6 c: |% d2 ~( z& u: S0 |5 n: N. T
8. Unit 10.7 Proteoglycan Isolation and Analysis
+ v- Y& g% Q0 W6 Z+ C: h' h* @9. Unit 10.8 Matrix Metalloproteinases8 Y6 s8 V0 }- S/ R W7 D
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
; s9 ~% o" ]& n6 | b11. Unit 10.10 Purification and Analysis of Thrombospondin-10 ^0 c# i6 ^; F2 ^
12. Unit 10.11 Purification of SPARC/Osteonectin
6 B2 H0 x1 N, v) d/ _0 Q# P a13. Unit 10.12 Analysis of Fibronectin Matrix Assembly2 ^, ]) L' w2 K$ w% g$ Y
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly+ C4 h( K; \3 g/ U/ J8 `
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor% G$ w7 T, t( {" X: e+ |
Xenografts
+ Y7 c3 ?; N* }% Y! q; I16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
- ~1 e% C m B% Y' v17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
+ ?, |+ u1 L, J" ]2 T* |" ]- X# H+ e18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
7 C1 S5 y8 {; l8 _4 H+ kCell Migration* g# d7 W3 w5 Z* {. y
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix) m( \' }8 M$ ?- I8 x2 i
13. Chapter 11 In Vitro Reconstitution4 X7 D$ V+ `& s/ a+ i
1. Introduction
6 o7 }& U$ [/ c3 P! H6 `2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems& t5 H4 p% N2 A0 s% K! b
3. Unit 11.2 In Vitro Translation" m" L- P5 T5 Q# O
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
U& S3 z- ?9 B5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes" b J' a7 U: \5 H
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
6 r X/ x: N8 W" I8 ?5 I: ~7. Unit 11.6 In Vitro Transcription
- [) F/ t. i; k8 k3 X: r) C8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
3 v$ R) Z. ~! V3 H9. Unit 11.8 In Vitro Translation Using HeLa Extract
: G, k2 r0 A) T3 l! t: i% j4 k10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
1 A, D0 t: e5 P2 V' k: ]/ @0 C11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts* K, W1 C' j: C" z1 I) L
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
6 d0 W& F0 w$ M! d13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
2 ?( k; ?9 X! c5 T: d& k( U14. Unit 11.13 Mitotic Spindle Assembly In Vitro: n) U) l5 `+ v8 _4 O# M7 b- m
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
' Z/ [& \9 a: C* f; k* _16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
P: G! o" Y( X6 y' L6 C17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
" u3 N F* J& f" X- f18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts m4 e: R& h, e8 ^- f
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells, F, L) Y, A6 _7 y( D6 S6 X0 D
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import6 i% L, y1 [" b, V& |- Z0 z1 b
14. Chapter 12 Cell Motility0 a; \6 L4 F+ w* B" \+ x* f
1. Introduction
8 r7 f7 e* W, W/ o8 h, ?2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells$ l9 C k$ ~; c
3. Unit 12.2 Invasion Assays6 N' b0 m7 X6 H; @3 X4 H# a. M; f
4. Unit 12.3 Cell Traction" o9 B1 p+ }# ]9 G
5. Unit 12.4 Cell Wound Assays9 o( x! A) Q9 B3 }5 K* H
6. Unit 12.5 Dictyostelium Cell Dynamics
7 B7 L$ P7 Z+ w7 w9 O, K7 X$ ~7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
0 I2 [* D0 W4 y+ [: w8. Unit 12.7 Actin-Based Motility Assay s" a) j5 t. U5 C" @8 ]
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP2 Y6 B E: O* @# B2 q4 K7 k
15. Chapter 13 Organelle Motility
3 ~, Q0 L7 y3 D4 ^$ u) i( @1. Introduction
- Y/ @3 s/ ~! s/ ~2. Unit 13.1 Microtubule/Organelle Motility Assays) ~) e" U/ o2 s+ G$ z% \3 [# K
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
* e% _4 j7 W' c$ j& p4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP1 h, U! _5 P8 {/ n( ~
5. Unit 13.4 Movement of Nuclei
9 k* K0 W- G1 ?6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
- s8 |1 R9 X+ J& H0 z7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
- W7 A% N: e9 h, C2 M3 P, ]6 R16. Chapter 14 Signal Transduction: Protein Phosphorylation
" n. g# k# c. d: ], c/ {1. Introduction: l0 O) N& a O: _7 a% Y P
2. Unit 14.1 Overview of Protein Phosphorylation1 y9 Y/ r5 h* a
3. Unit 14.2 Immunological Detection of Phosphorylation
" b! X* N6 f7 H e' r4. Unit 14.3 The Detection of MAPK Signaling
) E y/ w' S! h( }* L5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation5 c4 D% P$ E7 g, {
6. Unit 14.5 Phosphoamino Acid Analysis
9 I# d V; {8 |7. Unit 14.6 Determination of Akt/PKB Signaling3 H; w( p$ f: u r' @' r, d7 O
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
/ _, }% h2 J- S4 B8 E5 \, ^9. Unit 14.8 Rho GTPase Activation Assays6 ?8 q' _( M0 {% E. h+ ^# j
10. Unit 14.9 In Vitro GEF and GAP Assays
+ G' `& q0 }0 ~9 V7 k( q11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance1 t; V" s9 q: W* s
Energy Transfer (FRET)2 H$ \8 z' Y, L4 `0 U% R& _
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells \0 x2 p7 t* r x' i0 }- M
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
3 c Y/ x+ d0 V17. Chapter 15 Protein Trafficking
+ ^' C( H. Z5 H! w( G1. Introduction
0 l9 D1 Z0 H4 I# z! f2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways& k( H6 ~# h0 U% a2 r8 c
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking* `( ~! W& D: ^3 y/ K/ R) c* }3 f8 y4 G
4. Unit 15.3 Endocytosis: Biochemical Analyses
) Y1 ^% d# p, W1 o5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
~& O) c) Z* W' o# \4 D; O6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells2 ^2 n4 q# D3 c$ \7 e' N* b8 t* `
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
9 b, x c+ Y: u" M$ ~! B0 @8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
( @" F3 S$ u7 S9. Unit 15.8 Analysis of Protein Transport to Lysosomes
5 e9 Q$ K3 d! U10. Unit 15.9 Studies of the Ubiquitin Proteasome System
: m7 C* h: T. J- ]9 T7 S5 v11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network" }7 r: }6 g, ^* E. H+ k) Z
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
5 a5 L7 }/ J, Q; n13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells" M9 R9 A8 \4 `
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
2 {4 ~# o. \ ~* _* F15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and2 U9 E5 r1 ^! L" |' V5 G
EGF Receptor5 l) w% z! |9 A0 Y; e" j0 x
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
/ w; }! t$ H, X; X9 {$ B18. Chapter 16 Antibodies as Cell Biological Tools% c ?+ W Z0 z. `) B& B# N
1. Introduction
( z0 U6 A, k( A9 V2 h2. Unit 16.1 Production of Monoclonal Antibodies
$ R5 L8 J" {- @% K3. Unit 16.2 Production of Polyclonal Antisera2 `, A) D# U2 o) J0 W9 ^
4. Unit 16.3 Purification of Immunoglobulin G7 k, W0 ^$ S) X3 k2 i O
5. Unit 16.4 Fragmentation of Immunoglobulin G1 e5 I& I1 ]8 b0 X, _+ P9 E+ a
6. Unit 16.5 Antibody Conjugates for Cell Biology
/ ^1 f9 L& k |% Z# k7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
' {& B, M; I, b- a# ~8 v, ^19. Chapter 17 Macromolecular Interactions in Cells- V" k, [! q$ n' @# g7 F
1. Introduction
5 z2 T2 L1 r/ \2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)6 N) H& a3 S# D! Y) n! { q% Y
Microscopy T F G$ c0 P4 H2 q6 Y' m3 J
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
# r' E$ n# p2 _9 J. E. T4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins2 E/ O' J6 z! J8 K( |) o
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries9 |% x2 G) D+ n5 \ ^/ G# `( `2 A
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry) n& W3 x0 V! k* L) E
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors8 U5 j ?( ?) f# G1 K
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific! H% A- w. H: a6 f/ t5 x, `
Genomic Sequences In Vivo( Z! E/ i$ L( s: W* y u
9. Unit 17.8 Isothermal Titration Calorimetry
* Y1 W7 p5 X2 i7 ?+ I10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells9 u: u3 Y8 H6 n! j% J7 Z
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking l5 w* A! Y; d d; f) Z3 E) o
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
7 a5 W1 z/ y6 W9 l2 N9 e: f( i) PInteractions (RFAP) in Live Bacterial Cells
& E' F; `+ m3 j. n! [20. Chapter 18 Cellular Aging and Death
! d3 u: _2 W3 W' Z/ D1. Introduction7 U4 _3 z/ B5 F! u3 M
2. Unit 18.1 Current Concepts in Cell Death
; _. c2 S6 _( m# i) c3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
& {& a7 n. p- ^, j, [3 o4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
# d1 d7 Y/ G7 c# g1 D! _5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
9 _* V/ O& R/ p8 A. v' _6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
( ]( n' k" v# U! u7. Unit 18.6 Analysis of Telomeres and Telomerase1 L% z$ X9 T; N7 v$ A1 r" V
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of) B9 R! W' S3 r# R+ C7 L
Poly(ADP-Ribose) Polymerase6 i* F* H5 r, J( a8 z, ^
9. Unit 18.8 Flow Cytometry of Apoptosis; P9 r3 l% h' v( S, `5 t$ u
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
) a+ s2 N) j' r! Y) RAssay' i! Z5 G8 m0 W" N
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
! j8 q& A4 c, r7 c& O4 f21. Chapter 19 Whole Organism and Tissue Analysis) e. x- j W1 o, p4 w+ p
1. Introduction4 [, a9 K# d; c5 o% C; K
2. Unit 19.1 Overview of Metastasis Assays1 P O* X* y- P" z
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis$ r$ S- x @; f r
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
4 `- R! M5 y5 T: C, _Expression After High-Fidelity T7-Based RNA Amplification
' M( |, s! ^& j, @/ h" j& S+ p5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
' w o9 y) K, ^3 A/ |; X6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
/ P" p6 l4 Z7 ?! b, ?7 A4 P0 b) Z7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo# P: m+ L( G$ J' H% G, \
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo* D& ~& V1 N- s
9. Unit 19.8 Embryonic Organ Culture
C; X4 L# s( N" H10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
, ^! r A7 s! p2 W7 Y+ r; e) R11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
* `1 Q0 V! Y5 q12. Unit 19.11 Generation of Transgenic Mice/ _& u& i3 {: `7 Q3 g
13. Unit 19.12 Overview: Generation of Gene Knockout Mice
% L' \- f$ q0 C3 w/ A2 T1 R& W14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production+ ^- U: R. ?; \
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection- _' P, H7 {' K+ v$ r U
22. Chapter 20 Expression and Introduction of Macromolecules into Cells/ B/ n' Q" j( s# z& {2 V
1. Introduction4 o4 i4 j; V* P
2. Unit 20.1 Direct Introduction of Molecules into Cells. J L: ~. t- a
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
/ A" r* y1 A' W5 j% d- d3 {) |4. Unit 20.3 Calcium Phosphate Transfection3 L' o& p1 w2 [- j
5. Unit 20.4 Transfection Using DEAE-Dextran
# X( s3 R, d1 a, e6. Unit 20.5 Transfection by Electroporation" j1 ^* g, x. B+ x1 Z% P
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
. Q" k: C4 i3 L4 r5 P$ ~( R: H8. Unit 20.7 Optimization of Transfection
* O" u1 q4 T7 X- u# @5 \9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
6 |; D1 g' J- B* ?# B! t5 U) t23. Chapter 21 Fluorescent Protein Technology
2 o( Q" n+ d0 C9 y1. Introduction
* J' V E+ i w: M6 n" _- A3 N% m2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells% \" c5 O' E9 m' y: J; c p
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)1 ]- L( P$ B. z$ b( w
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence% t3 Y) V: _% E
Complementation (BiFC) Analysis G1 ]; H" f+ @$ W" C4 z, l
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
$ V/ N- N/ r1 q; L6. Unit 21.5 The Fluorescent Protein Color Palette1 r! T1 `9 ?4 a3 W5 O) Z' U
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
- I& `* i3 @4 \0 f24. Chapter 22 Cell Biology of Chromosomes and Nuclei% e* v/ j# ?. V* g
1. Introduction
& I& y* B/ {9 n) z& C: Y1 S2 o2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis! D4 X& F2 t2 U; e4 v4 W
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples9 z9 k6 {: C( m0 a& C, x* b4 z8 y
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis; V" N2 I$ M5 z8 c7 \2 f/ X
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)2 u" G# p0 } K% ]7 j& n2 z w
6. Unit 22.5 Multi-Color FISH Techniques
: I( m6 y/ s/ ~$ F f! T7. Unit 22.6 Comparative Genomic Hybridization9 D7 h# M. o. x
8. Unit 22.7 Sister Chromatid Exchange3 m; t' L4 X7 k/ R6 @" k
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
+ c$ a8 N! l3 K9 K0 g9 A" Y9 b5 v5 T10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
7 {- ^8 ~6 X, n11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
2 O6 E3 |+ W' | d/ G2 i12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
- n! m) x# q+ k% p# {13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells# j+ ?' K0 {- p3 ^! n, s
14. Unit 22.13 Monitoring mRNA Export2 J1 c2 L% F& H3 ^' M
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
8 y+ W! _4 c9 PField Gel Electrophoresis
. ?2 i* @- \' G# f4 c3 @5 x/ l25. Chapter 23 Stem Cells4 q+ k. g D( a/ H" Z
1. Introduction) I$ J& o2 T: w3 o/ e3 H6 J
2. Unit 23.1 Stem Cells: An Overview
) a' c3 |) N Y8 l3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
' c' x! ?( j9 j. u* H! W9 sand Differentiation as Embryoid Bodies
( `! a! K- u! _( N. k: g$ s* S4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood) Y5 h* N: C" l. d/ D
Vessels/ x; B6 V, o% r
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
& o- J4 U0 j0 z1 \) R+ wAdipocytes& }" t9 b+ `& X z; E
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation) G: ~/ D o7 \* i) [
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells' q5 |1 U; Q( D M1 _
8. Unit 23.7 Neural Differentiation of Human ES Cells
2 M9 m. H% \2 n+ [- ~1 N& g$ L26. Chapter 24 Lipids
. `/ c4 p! K3 L+ W" u7 ^. r; l: R" R, l1. Introduction
3 M, Y' f0 Z! i1 ~4 w+ N8 u( {" [2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
3 N' H: Q4 w6 X' L, k7 l2 ]3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
2 l- u& P% ~: L# i- i4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film1 p; s% U- o; n
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
/ E/ G$ i$ m: I- `27. Chapter 25 Nanotechnology, F3 ^( `2 a" O2 X* }
1. Introduction
& S9 F' Z5 b) w }. e3 ?2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
8 F) e/ w+ |: Q' D3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
; a* s5 H" y8 ]% E/ n. @28. Chapter 26 Viruses( s; i- G' n3 `9 |
1. Introduction) T; A N0 P4 B+ a4 c$ s) n" l
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors7 s% ?7 a: e1 [7 x' R1 p* U3 F, I
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of' D3 \+ w; O; N T5 I, I
Cell Entry, s! g1 A0 I, B: g/ {4 i3 u
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection9 |) O( I% F n9 Z$ v- r7 H
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses9 W$ q# Y2 _2 j( v+ `1 h6 P
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
$ k: U+ Z; M6 Q: T1 h3 e29. Chapter 26 Lipids) \& D0 `1 j! |6 C( l5 @
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by: x9 q# F) t- G- u
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
3 v9 R1 o( p+ v: Z9 J# I30. Chapter 27 RNA-Based Methods in Cell Biology# E7 L# |/ T& O% ~& Y# i9 R
1. Introduction
" m. T2 i9 s2 P/ ]2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
; Y8 h8 X, l2 J7 s2 w# R! S6 M3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
* l$ ^* D W+ f1 \* q( }Rescue in Mammalian Cells
6 V# B8 c! a+ K: U; Y31. Appendix 1 Useful Information and Data8 r. H: L% M" a+ c+ @# q B+ M
1. 1A Useful Measurements and Data) L1 z" J2 w S5 y
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
! l( t( x6 w1 l6 i$ E1 ?9 J- X3. 1C Identification of Motifs in Protein Sequences! f X: n3 s' [! N* L0 q! z# s
4. 1D Safe Use of Radioisotopes
; }: C, @: e B' {8 X5. 1E Absorption and Emission Maxima for Common Fluorophores
' Y# t) F8 e' q5 W$ o6. 1F Importing Biological Materials
9 M/ W. P* Y7 J X+ j0 m; i9 K7. 1G Centrifuges and Rotors2 T. Q! w, n( [3 b/ k/ T
8. 1H Internet Basics for Biologists
( d, a3 Q8 I8 i& Y i! E32. Appendix 2 Laboratory Stock Solutions and Equipment
4 ]1 [( \/ {* o1. 2A Common Stock Solutions, Buffers, and Media
! J' i! o- h& b3 h$ j2 \2. 2B Medium Formulations1 F0 M, A5 H7 n2 u4 B) h& |7 `5 ?
3. 2C Standard Laboratory Equipment
( v7 i" P5 R( i7 l$ b; z33. Appendix 3 Commonly Used Techniques
2 w" |6 s; l' c6 l7 h, ^1. 3A Molecular Biology Techniques% I; G& w/ A: C7 H1 L5 f& d- K
2. 3B Spectrophotometric Determination of Protein Concentration2 g+ M. w1 r& U9 x) o
3. 3C Dialysis and Concentration of Protein Solutions
H5 Q0 r; r8 Q, c/ L7 J2 i8 o! {4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy3 T% J5 F( T$ X S& K6 y5 c# F
5. 3E Silanizing Glassware
5 b2 ?0 y; e# n6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
+ a$ }7 R; ~6 q v7. 3G Micro RT-PCR
! F, l1 G0 ] E' q" J. e2 |5 c- v8. 3H The Colorimetric Detection and Quantitation of Total Protein
. z# i" ]* G/ K. |34. Appendix Suppliers
1 s" M1 d# d& [7 D- c1. Selected Suppliers of Reagents and Equipment; w0 V L$ [, o( o
1 _3 Y; C1 H( V4 D) q @3 i, [# ] |
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