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' z' S( g* F; v3 i2 aCurrent Protocols in Cell Biology 2010年完整版 5483页
4 J7 o( I' O3 m9 J6 g
1 ~) K! E8 U8 j( ^( s" ?9 ^Online ISBN: 97804711430319 d, T+ I& e0 s) `$ W3 a/ ]( P E
DOI: 10.1002/0471143030
9 _" Z9 o/ ]+ }9 r3 E& M( Q+ \/ H
Table of Contents
9 I9 [" R. h' g! f* G+ p1. Preface1 u' E, M( @( R& ?" h
2. Foreword
5 y0 z; |8 l1 P- o: A3 c& U3. Chapter 1 Cell Culture
% Q' q: X! \+ Y* B0 u' B1. Introduction
* c9 _# K: K' z0 _% Y' @& |+ s2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
- K# W: N% v3 I* O3. Unit 1.2 Media for Culture of Mammalian Cells+ H1 b: a; J5 C M8 D" Z# `
4. Unit 1.3 Aseptic Technique for Cell Culture
7 P' I$ E. e& g* E& T* D5. Unit 1.4 Sterilization and Filtration$ a2 Y0 E) g1 e
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures* N0 w* D, J4 f& I% [* B2 d
7. Unit 1.6 Media and Culture of Yeast6 n8 e+ {& W, m) F. n9 `
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging! X5 k3 p. k0 ^7 _" Y
4. Chapter 2 Preparation and Isolation of Cells
, m; |. G% z( s7 t+ x1. Introduction
& O8 V. I* X5 F+ Z# K% \8 q2. Unit 2.1 Establishment of Fibroblast Cultures7 P) z6 ~* k. q) }& G& |" k) ?
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
; M- [. s+ [/ G) e4 P9 i4. Unit 2.3 Preparation of Endothelial Cells
) e7 t) I* ^4 n5 K5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation/ C6 m6 T: @$ F# q8 F
6. Unit 2.5 Laser Capture Microdissection7 C' l& F+ [# i9 e
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures0 S0 B: S1 k% n7 n! H
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells- Z9 q* D1 u; _7 D- W% X
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
( y) `# a8 ^' [1 U Z( ^1 M4 s1. Introduction
% k+ d5 `. h# N9 Z7 a2. Introduction
4 u# E Q: b& p+ W* {3. Unit 3.1 Overview of Cell Fractionation
H% b) D6 z2 [8 {% Y4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
7 e7 d" a8 m/ i- R1 g9 t5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation: C) v! @0 E1 L
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation* a+ p: D2 N2 J' L/ I- I- s- _
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
3 d3 ^9 G6 w6 P X; [Centrifugation7 b$ `4 A' g7 H3 l
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient& I* H& @* b% c) ?" {
Centrifugation
/ l" I1 b6 h5 ]6 |' n. |* n9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae- L$ z3 i. F/ c/ d$ a
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae& R8 u- E: T* Y8 ]% P6 |
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient! A& e% B3 H1 ^8 Y5 B! N0 X. N9 F; g
Centrifugation4 r$ R) a! s2 `3 u+ ?' `9 T3 r
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues' m6 d) L: ?8 `# i2 n; P6 e. C! V
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
- t% H3 t6 R. ]6 ?7 t, D- V# @14. Unit 3.12 Isolation of Synaptic Vesicles" L* n: d( w6 {
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
3 p+ f5 r ]% Y" b* U& a* w9 Q16. Unit 3.14 Isolation of Melanosomes
! I2 } ?& D M. p5 S7 F% T* U17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation1 E6 C% J5 c8 B( u* { C; d
18. Unit 3.16 Isolation of Mast Cell Granules# d- w Z+ ~& R0 u
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster X( c. E5 u" A. R; r; W1 d, ]) f
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
: E( A( o! g. N% `4 D7 L6 O21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons% L# K4 m4 c, e3 S4 B) Y) \- g
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles! j* Z& n! V/ K; P! s" i
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
$ ~. A0 N# \& P1 g4 r5 D# Y24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
% Z6 Z* V5 N* _2 h5 G/ H2 ^" x' a% jFluids' i5 Y, G4 [4 Q5 m7 J! e
25. Unit 3.23 Isolation of Intermediate Filaments
' X+ M% J4 m/ C6 J8 b, e: D26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle& o7 }4 }- D% t6 \' h: ?7 }) h
27. Unit 3.25 Isolation of Myelin) J7 t- ^' l! S( T9 |4 O- @
28. Unit 3.26 Isolation of Renal Brush Borders# p/ p) {: D8 C6 c4 X1 p2 A/ S
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
0 u! Z6 j0 e4 ]. FFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts9 K* _' v& \! Z# T* B
30. Unit 3.28 Isolation of Amyloplasts$ g R: T3 h6 X9 K9 Z
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
" y: k# ]6 Q1 s8 b5 C" ~32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic# d, Z/ S3 S4 Q# L
Centrifugation
! `" X1 `* X4 \! ?3 E33. Unit 3.31 Isolation of Neuromelanin Granules7 n( ~0 g. D( @' |, g- D
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and3 i5 b9 \9 |" [% Y9 o. t+ H
Density Gradient Centrifugation( Z; j5 J2 ]; a! v% i- e u2 n( }
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
9 ~5 ~. y4 R7 _- R0 [- U36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation! {- _4 Q) a/ [ q9 u5 n6 m$ a
37. Unit 3.35 Isolation of Platelet Granules7 a4 f" P8 @4 U6 I% [* ~( s
38. Unit 3.36 Isolation of Nucleoli: s( p# O- b% g5 H7 ^& `; u( S" e6 h
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
* F5 g" A# }* J3 y. q40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates# s- s+ ~ i! s6 q! F
41. Unit 3.39 Isolation of Chromaffin Granules1 F: b* j( Y* d6 s) S1 S" a0 P
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
0 q2 R* ]5 K/ P6. Chapter 4 Microscopy
2 _- _: I' }5 n( n1. Introduction
& |+ Z P0 e! V1 [: U) V, c2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
# |; E9 |. N: M; A0 y) }3. Unit 4.2 Fluorescence Microscopy2 \9 w6 l i2 g5 q0 t3 n
4. Unit 4.3 Immunofluorescence Staining
) o, F- t7 M0 [# v: e% g5 S5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
" J4 i, p" S( U; Q) a6. Unit 4.5 Basic Confocal Microscopy/ M$ `% e* k' C+ A! t
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
! E5 y$ i. i n# W+ j$ l8. Unit 4.7 Cryo-Immunogold Electron Microscopy
: G0 e( B9 X( ^" |2 y: |9. Unit 4.8 Correlative Video Light/Electron Microscopy. e8 Q/ w5 O/ _' v7 A. M
10. Unit 4.9 Polarization Microscopy
4 o, l' o/ G7 Q3 g3 C11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
6 k; Y+ A2 ^: D/ n) K1 R8 j9 B12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues) S' e0 E. W! I! v/ w& t2 ?+ }, J3 A
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
9 Y4 L7 w) M( H9 Y3 c5 }Events- E0 H) o. \' ~0 b/ r( O1 F$ R9 ]
14. Unit 4.13 Fluorescent Labeling of Yeast7 Y" L3 g; W2 N* i7 u
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
# }/ H& T6 v v+ _& U& _6 _16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy( s7 x" }! d8 `' F5 b+ N
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
W3 e4 m" a4 Q$ h5 Uin Endocytosis
" D' G* `; |! O7 x5 w. q# W/ O18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology" m; B) n1 R# a5 Q
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development' @% a8 E" I, @0 V
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
! X; y- G. J9 s' Q, Q. I! ?21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
0 S' r6 Q) w- ]/ [8 Q7 ^1 r u22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes5 J3 Q* H. z* J) b. P/ L. z
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
" t% A% Y" I( u2 m9 E24. Unit 4.23 Interference Reflection Microscopy% z% r0 F+ e9 a; S
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
$ y( h) M* M0 o& n7 t26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches3 A: Z7 t# w6 v
27. Unit 4A Organelle Atlas: Appendix to Chapter 44 P- t7 f9 ?% v" \
7. Chapter 5 Characterization of Cellular Proteins6 _4 C, W& N5 u: y* x% a
1. Introduction/ k% a9 d/ o( y
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells# Q4 T' g3 B. h$ E, v
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
" W! v, |5 @* L% y4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
( ]. g; F4 I: ?$ {+ |- k5. Unit 5.4 Analysis of the Association of Proteins with Membranes
* f+ e$ [5 h2 [# |+ _6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
- g x) _" b C O7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass$ p7 l2 y+ F7 \4 R/ r5 E% `
Spectrometry, }9 b( s! j+ U. C
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening& a, ]2 M& j7 m V
Applications
8 y# X4 `' y! I+ r2 T8. Chapter 6 Electrophoresis and Immunoblotting
. H4 {. C+ A4 ^0 u: {* ?- U& a( Y1. Introduction, y+ ]( ]: n q+ G" U0 i2 T
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins! |3 Z8 K9 e2 d- z0 q: Y! ^
3. Unit 6.2 Immunoblotting and Immunodetection
; C( V; q7 H5 Q) m, l4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots% ^. u | S) M% {& K7 U7 M
5. Unit 6.4 Two-Dimensional Gel Electrophoresis; m( g" B! h% N5 D+ `
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions0 H6 _0 F( l7 { P( u5 T) ^6 {
7. Unit 6.6 Staining Proteins in Gels/ [! M+ f! L9 X/ ~ \5 t6 k
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins( [. |& h( [3 A6 |+ }% _
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots( l2 P$ V# @4 m! E# e' z
10. Unit 6.9 Digital Electrophoresis Analysis( _7 b- Y+ z( c" D5 f- |4 D w
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis+ S: k7 E5 s- I6 X+ t- n
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of B2 g2 a" u5 r4 g; ^4 m8 m7 L
Single Cell Gel Electrophoresis (Comet Assay)+ |" w7 d$ E1 e$ x) X' }
9. Chapter 7 Protein Labeling and Immunoprecipitation1 f( I% v# m P A( B
1. Introduction8 i( H: m& ?: `, }1 G0 ^6 C; V
2. Unit 7.1 Metabolic Labeling with Amino Acids3 o7 @8 @ ^9 c, e8 j8 M k
3. Unit 7.2 Immunoprecipitation+ u. L* S; y/ s+ ~1 h$ _+ j
4. Unit 7.3 Metabolic Labeling with Sulfate
0 a" U& N% J: u3 j2 u5. Unit 7.4 Metabolic Labeling with Fatty Acids
! L- e& b% j2 B8 ]6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
: |# }7 E! t+ G/ `3 ^, S7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins9 j1 U! C: o, L7 R8 t! r0 x
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins Q* |# f+ Q/ D) [* t6 o
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars2 G) r, G7 D4 [' s0 d
10. Unit 7.9 Analysis of Oxidative Modification of Proteins7 r+ m1 L) @" s Q4 B
11. Unit 7.10 Radioiodination of Cellular Proteins
8 N( J5 `5 Y+ s0 u+ o10. Chapter 8 Cell Cycle Analysis
% s: x; b: { x! Q, r% n/ l: n+ Z+ i1. Introduction+ r/ {) a s% I0 F0 |* x7 L
2. Unit 8.1 Overview of the Cell Cycle O) l- V* L8 b: x( O: E
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
9 E& `. s ^; I4 F% J4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle) ~6 Q# z' x, z; _. U
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry0 c% ~$ d( G4 L% t3 Z: K3 y' Y
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
" I- G4 V0 R2 }7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry1 y2 _2 l" g2 ^+ h
11. Chapter 9 Cell Adhesion
, o9 U6 M8 `. S0 r5 O+ J" |/ T1. Introduction& v# E w9 a7 Z7 _$ j! T. Z
2. Unit 9.1 Cell-Substrate Adhesion Assays$ D$ q6 M6 i: Y2 G l0 g) b- g' _
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
5 M( m6 Q' h8 j4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
1 F: a' q1 `; c5. Unit 9.4 Analyzing Integrin-Dependent Adhesion% D8 F) U/ g) s
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules) h# K, c. z& g+ m
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions& I9 P7 A' E! J/ c4 u1 K9 X
12. Chapter 10 Extracellular Matrix
U2 U1 m8 Z, l( T9 G1. Introduction" X* B4 l$ w5 g/ z( {+ k9 c
2. Unit 10.1 Overview of Extracellular Matrix/ o6 M7 g0 t# s* {
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors& R0 b2 a. _. g; I' {3 Z
4. Unit 10.3 Preparation of Gelled Substrates2 A' j0 a* O7 U8 s) q* P. N
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR99 `* U' ]. w/ z8 B8 \4 ?" q
Endodermal Cells
) r; _( A( c2 A5 H8 ?6. Unit 10.5 Purification of Fibronectin9 b! B: u# X2 A O. U6 z: }; Z; z
7. Unit 10.6 Purification of Vitronectin& |- N, J$ F% }" ~: r) d0 N
8. Unit 10.7 Proteoglycan Isolation and Analysis6 `( ~1 k9 M9 D* l' g& T
9. Unit 10.8 Matrix Metalloproteinases- }) {7 T e& p/ N5 f
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts( i% z+ k3 g! T9 X
11. Unit 10.10 Purification and Analysis of Thrombospondin-1) D, _* H8 b" N1 V/ ]
12. Unit 10.11 Purification of SPARC/Osteonectin
$ H# K" K$ J+ E% ]3 K/ Z13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
5 {& c1 l4 L1 Q7 i" |14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
+ o* H. w! n4 C. H5 n7 j15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
% f; c- U/ u9 S& b7 uXenografts4 S; O! Q0 X/ u1 C" ^; L
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
& ^0 C% b: \1 O: d) H5 ], @4 |17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
2 \) z2 \( N" ~1 U/ X18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
, r, G n# U. K) w! VCell Migration" Q, R' C% \& P( F2 p8 H3 Z
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix. b$ `' e, T1 P7 Q
13. Chapter 11 In Vitro Reconstitution1 ^6 Z5 u6 ?& U9 r$ e/ S" L& N, ~
1. Introduction8 d4 F" ? X5 C" g# B9 \
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems) J! |: z+ p0 Y3 N6 N8 n, T
3. Unit 11.2 In Vitro Translation9 F7 |) x8 \2 R& p. z+ x0 [
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells) t# C2 j" u# e, t
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
9 P0 {" E+ ? Z* w( q6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
" z3 g/ O* F V8 a% E& f7. Unit 11.6 In Vitro Transcription
6 a+ a- W0 @3 E, L+ h9 _7 m8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
4 u* r% b6 n3 i0 s! j* M; X' \9. Unit 11.8 In Vitro Translation Using HeLa Extract. r' n& a" X* L2 K
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems8 T, B8 [" \* d9 C. P! C
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
0 E& W$ w8 g! _* M12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts- |" x8 j4 C, { }, j1 R
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts) c+ Q7 v' L! `- Z/ d
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
3 W/ _$ v" Q. l. K* p3 U15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
( B+ I. @% k# Q( @. c4 j. S16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import& j% X. S! v* A w0 m3 }
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import# g* b6 i" a$ d. u( `8 D" \# X7 Z% B
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts7 g9 z6 R& R; y2 k8 T0 ]
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells8 n4 y+ `, B$ s3 Y6 H
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import4 ?5 H3 z! X6 C# ]& J/ M3 K2 e3 U
14. Chapter 12 Cell Motility
( T9 F# `! T4 r9 ?1. Introduction) J* t3 F9 p# ?1 R4 \, l
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
. A8 ]) ^& Q4 W1 e* g1 V; E3. Unit 12.2 Invasion Assays; V6 E& w `* T
4. Unit 12.3 Cell Traction
2 l9 ~& M% T O, Y' Q' u0 V5. Unit 12.4 Cell Wound Assays) R' ^9 c" l3 a
6. Unit 12.5 Dictyostelium Cell Dynamics
5 |& _: T9 y: N2 z0 T7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
( m+ C& B3 }" a2 s9 @# T( j1 D G8. Unit 12.7 Actin-Based Motility Assay# W0 E0 T2 C; R$ D8 l1 ~8 Q
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP! K# l3 F2 }+ R& U5 U! ^
15. Chapter 13 Organelle Motility
, x8 m9 a" J2 u* F+ e4 j( r1. Introduction
2 G+ _( @7 t/ R4 c1 I* c4 D$ E$ G6 `2. Unit 13.1 Microtubule/Organelle Motility Assays
& N. w! r5 R* ]: I1 q* W x) e3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin1 j; @1 }' ~, H2 f) \ v( F
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
- E; t3 U5 F3 l: Y7 ?7 M6 s7 s* S5. Unit 13.4 Movement of Nuclei
A5 W% V8 n4 b2 ?6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching, y- ?8 I9 f; z8 _
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly4 {' ]% y9 p- _2 m
16. Chapter 14 Signal Transduction: Protein Phosphorylation
2 p4 ^0 l: o' z3 I* x1. Introduction
0 a5 N! x0 c- g* O T; j- E- d2. Unit 14.1 Overview of Protein Phosphorylation# H) H$ \9 Z+ \; w X
3. Unit 14.2 Immunological Detection of Phosphorylation
" ? X% C% w0 {- ~: `4. Unit 14.3 The Detection of MAPK Signaling" }9 N7 x: q# L0 f7 _3 T
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
" Y. j5 e2 w4 p- P6 o8 m" }6. Unit 14.5 Phosphoamino Acid Analysis. K2 S5 s8 }7 H9 I7 _
7. Unit 14.6 Determination of Akt/PKB Signaling
7 X8 b% Z% E! I+ T) f8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events$ i- ]) F. F- K. i5 ?% G# }& p6 C
9. Unit 14.8 Rho GTPase Activation Assays7 Z5 W7 s8 Q* ~& T3 R b6 S
10. Unit 14.9 In Vitro GEF and GAP Assays
' Q) n, X, H, t% Q11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance; u) n5 Q# G6 ~$ Q2 X
Energy Transfer (FRET)2 M8 `3 |5 Z2 D# O" `, E0 r
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells2 Y! N8 v8 _1 n9 u1 C9 a& l( ]) ^
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells- N" t, v# N# P
17. Chapter 15 Protein Trafficking; a/ {+ @0 H6 J4 C% E6 K
1. Introduction
, q# Q I1 j1 q l! | M2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
* f" ?1 z: K+ M1 b3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
9 Q! X- V) [6 f( Y# B( `" l9 e4. Unit 15.3 Endocytosis: Biochemical Analyses/ }5 U7 o; m6 d/ f9 m2 x# O2 e
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane5 i8 u9 ^7 X3 s) O* Z" @
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells N) S! P. F. R) ?: |2 H5 z6 @
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
+ D0 G# ]5 W5 I u+ e8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation5 q) R. Z6 S* i, J9 k8 M2 k" G
9. Unit 15.8 Analysis of Protein Transport to Lysosomes5 N6 y$ V+ Q' ]8 v1 }: |
10. Unit 15.9 Studies of the Ubiquitin Proteasome System' X& s$ x% A1 d4 n/ ?* O
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
2 X4 A+ j @ G! l# \12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules7 V0 u# N6 I% o V" g1 b
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells; J& {" W% o7 A0 |* Y4 o3 ~
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging1 U' {- U% b2 z U' G9 Z
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and" d6 ^6 s' J. T- B* }5 Q
EGF Receptor
; G5 f V, {( d, v, x4 t16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
# x8 J4 T+ G8 i c* ^8 Y% S s! U7 V18. Chapter 16 Antibodies as Cell Biological Tools! D6 R+ Z! ^, r0 ?" O2 h0 c. _8 K
1. Introduction
" C# R! a+ q8 f% |% X2 p2. Unit 16.1 Production of Monoclonal Antibodies2 h5 r j: @; e4 i- m, R
3. Unit 16.2 Production of Polyclonal Antisera& l: s( K, o& ^2 c8 H
4. Unit 16.3 Purification of Immunoglobulin G) m# z2 G$ {1 ^" I# Q
5. Unit 16.4 Fragmentation of Immunoglobulin G
9 }* I- m$ U0 S: u& K4 {6. Unit 16.5 Antibody Conjugates for Cell Biology9 D9 s d: C0 v+ J7 O6 K8 K4 ?# ^4 H
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides0 i. A3 W* {8 U$ X4 j
19. Chapter 17 Macromolecular Interactions in Cells9 z7 p' W0 }5 _
1. Introduction) ?' O$ z4 |1 E8 W1 y! L; b
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
& r! h' {" w9 ^6 _Microscopy3 {( Q) S' k4 R7 p3 l" S
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
, [9 `& K4 {8 r: u2 [4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins% m" i& `# x. V+ F9 k' `
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries" R' j+ K8 d# s( z Z
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry h* p3 v$ G8 U8 d
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors+ q) D0 O# f! I3 X0 z
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
[3 l/ J% y6 A# M t$ rGenomic Sequences In Vivo2 m) P5 E( f; Z; S) L
9. Unit 17.8 Isothermal Titration Calorimetry
% V5 G, f3 w& j& e10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
) w1 B# Y- _2 a$ @11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking& U1 j0 k* m& A3 N! a' U# f
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
' P* O0 w; ~& U o3 D8 jInteractions (RFAP) in Live Bacterial Cells" w8 u/ `# n) j k5 ~* ^
20. Chapter 18 Cellular Aging and Death( L: }+ |* ?& Z" E
1. Introduction& H& X, S% c. J7 ^0 I. o; w& v% s
2. Unit 18.1 Current Concepts in Cell Death
7 T9 ^: X0 | ]2 E; M. Q* L- I# u3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
" Z5 O2 Z+ |4 E* ^4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
) M7 m( n* N- s8 `( J5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
+ F0 Z' X6 w+ m6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death! y& C& x4 A1 x; E
7. Unit 18.6 Analysis of Telomeres and Telomerase" w: _' l% a4 F! ~4 t/ p; ~; ^
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of3 k' M5 P* ]' c; R) e7 u4 r' c
Poly(ADP-Ribose) Polymerase
0 F* S! n. y4 ?, x# W9. Unit 18.8 Flow Cytometry of Apoptosis) @% m% W' W' ~, t; [/ D) O
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
5 T. D; V l% A) ^, ^Assay
: |$ s9 J! {: C0 m11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
( q/ o- |8 e: B3 o0 n21. Chapter 19 Whole Organism and Tissue Analysis
% v2 N# v1 B8 v% x* H$ t$ `1. Introduction
! x! J& o7 d2 Z) ~; w4 v9 I% J7 d2. Unit 19.1 Overview of Metastasis Assays7 @, V# L# _& O/ ]& D; T: ^
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis/ J; Z/ X4 n/ v, m2 z+ q
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
i7 j2 V9 b# y% L0 P% @7 f+ @6 GExpression After High-Fidelity T7-Based RNA Amplification) \, V. |# p. U& p9 F
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
8 \- P8 n7 }( Y4 I+ x0 x0 B, d6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model" t- e# Q1 U9 B5 ~) ]. M0 X
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
7 C' _4 c" n+ I: G3 A) i8. Unit 19.7 Imaging Tumor Cell Movement In Vivo' C8 X8 P+ p% h
9. Unit 19.8 Embryonic Organ Culture$ ?' e* L8 _) j4 `
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin) U$ U/ \) b' L/ P( l7 O4 \
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
1 m& C! v' k9 k! Q12. Unit 19.11 Generation of Transgenic Mice
* K( y) u: a' `7 C/ w- r13. Unit 19.12 Overview: Generation of Gene Knockout Mice1 G" S. ~. w9 B& d* w4 ]
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production* M2 W! J$ m# V& U* ?$ k+ C
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection. D! Y5 D& j8 @+ b( z' P8 h
22. Chapter 20 Expression and Introduction of Macromolecules into Cells' Z# Z0 \( G3 b H" \
1. Introduction
! m: [- e' L* b c2. Unit 20.1 Direct Introduction of Molecules into Cells
/ O* E3 i3 N" K& A0 u3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
h- Z2 V1 {0 F* ?$ Q) l$ _4. Unit 20.3 Calcium Phosphate Transfection
& V @1 b2 \. H/ W5. Unit 20.4 Transfection Using DEAE-Dextran9 V3 w& s) M' @( _ v1 _
6. Unit 20.5 Transfection by Electroporation
+ t4 A" P w/ l9 H7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
! j1 J% y" b9 Q V8. Unit 20.7 Optimization of Transfection
+ P3 i# J, [, t* U9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
/ l! ?: T2 b! C+ _5 a: m$ w4 K, s23. Chapter 21 Fluorescent Protein Technology
; r g5 t! t' T9 L: N* N" e, s1. Introduction
+ ^9 r" H( g3 Z* e. Q: x% m2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells# i3 j. L0 U1 ~; ]7 @
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)- c( ] W* h/ n4 S4 C
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence( I2 ?, Y- |; m* [* M/ I
Complementation (BiFC) Analysis7 v1 D8 z1 d8 a7 E
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
1 B2 E, \1 Y0 D# t8 O6. Unit 21.5 The Fluorescent Protein Color Palette0 R0 |7 R& T% O! r, N
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
7 Z; L' H/ S- N+ l% h24. Chapter 22 Cell Biology of Chromosomes and Nuclei
7 M. n) z( k+ G L$ l3 g( r6 [1. Introduction
* r! H8 H5 [, |0 w" I0 f# ^. d, J2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis$ p1 m, A, y) N9 e* b6 ]6 I5 t
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples/ ^% F1 L! Q$ e. r n
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
) @, F( \' x% o5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
4 c$ Y" T- L6 L. ]: u; q6. Unit 22.5 Multi-Color FISH Techniques' ?" O, E# v3 l5 C8 a* K) r
7. Unit 22.6 Comparative Genomic Hybridization
. x; f8 d7 C2 u8. Unit 22.7 Sister Chromatid Exchange
* i! U v1 Q$ l9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery' h+ n2 n, m1 G" N3 Y, e2 D/ f
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
( f; h% R6 m* `) t4 y* o11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
( ~% t! t; Q, Y" O12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly, Z4 }2 b+ j' J2 R# a9 O7 s. X7 f, m
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
9 i1 x! f5 \5 H14. Unit 22.13 Monitoring mRNA Export
: G, `' r8 Z* N' }& {8 L3 `15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
( J% t0 ?" o5 E% \9 {) q3 m* }5 xField Gel Electrophoresis
& x! c/ p5 D( \7 V+ ^% H25. Chapter 23 Stem Cells2 o. W6 B# @+ i
1. Introduction: {: M- I( N9 p" ~. P$ \% ]
2. Unit 23.1 Stem Cells: An Overview
3 U1 I5 V8 N! l) f3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture& A9 {- S2 W& C& z; Z. L
and Differentiation as Embryoid Bodies
6 ^8 G B- B" {: U0 z5 r4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood2 t3 f0 s0 ]' N6 _
Vessels) W5 x3 E- e8 a$ F# c
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into! s3 e% o* V3 T- F! l
Adipocytes" g! Y6 t3 K1 n" v# m) D
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
# c' p# R% a1 ?1 x& X2 K% j7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
# ]; q, `- ^% u4 S( z1 a7 h8. Unit 23.7 Neural Differentiation of Human ES Cells
, {- t) d+ @5 J5 h% z" x26. Chapter 24 Lipids
* S9 E5 `" @- u2 n# L1. Introduction
7 O/ E) I! d# i; z: `. ]3 f2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking! U5 o# x1 H# l. t" u! k( U9 g) _: t
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins4 y! K4 P3 \: ^/ w+ y1 F
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film7 B+ C( n3 f1 O7 v
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
4 d$ l, a+ M# J+ b. u27. Chapter 25 Nanotechnology
1 |& d4 r! g. Y6 {& \2 C1. Introduction; N! P8 C; G( X( T$ ]4 H/ q
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes: _" l2 z- Z4 I# \- T: [: ^
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering- i+ |# C1 E! @4 C
28. Chapter 26 Viruses) I) O4 [, f! `2 l. Q3 g
1. Introduction
$ K" F4 g" |. |2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
/ N, F9 Y+ V+ i9 P% R3 \2 o1 A3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of* h+ O: t" N! [, t2 e+ E
Cell Entry! A" r7 y1 ~, h4 a1 {! _; Y' T
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
, S7 _7 b. c+ }% j8 U5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses2 ^1 @$ I' @; a% R2 t
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
/ ]' I: o1 n y' [3 w( l5 j y29. Chapter 26 Lipids1 p9 [4 d8 _9 m& g. I$ a' f1 I
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by( i: c4 {( I# o/ N
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
; J6 A* v7 e" {# \. X30. Chapter 27 RNA-Based Methods in Cell Biology
3 `8 d# z/ e- a9 u$ n; Q1. Introduction3 M' R8 H! ?! t3 r4 B9 u- Y9 P
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
# v8 \8 Q# Z3 {5 I; P9 c3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible" X1 c9 o2 a# `" {( p
Rescue in Mammalian Cells
5 `% {4 f% c& r+ I/ u31. Appendix 1 Useful Information and Data1 B2 w- w- ]2 z: f
1. 1A Useful Measurements and Data, Z8 _; s0 `) w
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
! k: K8 ]1 K/ g9 ?3. 1C Identification of Motifs in Protein Sequences
: Z2 W5 E x5 B* ?6 \! y4. 1D Safe Use of Radioisotopes& w! C4 _. Y$ N* U- G- |: w2 g, N0 ?
5. 1E Absorption and Emission Maxima for Common Fluorophores
1 w- R5 R( l% s6. 1F Importing Biological Materials
5 r% r& l% h5 I D. X5 O: C L7. 1G Centrifuges and Rotors* ?. D2 w9 N3 S% ~" _
8. 1H Internet Basics for Biologists1 K# O" L. x4 `0 p& H& n1 C
32. Appendix 2 Laboratory Stock Solutions and Equipment" a: n9 w7 n1 Y8 w
1. 2A Common Stock Solutions, Buffers, and Media+ N: t4 H. {8 @5 P. }, ?5 e
2. 2B Medium Formulations
; F( V; t5 I9 _4 }, h6 r; T' X3. 2C Standard Laboratory Equipment( J/ w- Y! | J, D1 Q& B1 T
33. Appendix 3 Commonly Used Techniques: _% T3 k1 G$ O1 j' G# w
1. 3A Molecular Biology Techniques% x& v2 o4 r" e5 m8 T
2. 3B Spectrophotometric Determination of Protein Concentration
0 |6 E2 r. V3 E% u3. 3C Dialysis and Concentration of Protein Solutions
; K5 d( t9 a% I* P& C- `5 `4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
5 x+ M3 K1 r% p- {& _* F5. 3E Silanizing Glassware, z p% \; k# t2 f4 T/ j4 \7 u% E$ y
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization/ Z2 y8 d& c3 Y9 ]: ]
7. 3G Micro RT-PCR; P3 Q+ `- m' p( b) G* \' _
8. 3H The Colorimetric Detection and Quantitation of Total Protein4 x) ^# D- j4 c
34. Appendix Suppliers
/ O; O2 D- B q$ i w1. Selected Suppliers of Reagents and Equipment4 J2 b0 z' o. c" F
}" P7 J$ A+ n
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发表于 2011-3-8 15:53
