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1 A; I7 v$ a% |5 M0 MCurrent Protocols in Cell Biology 2010年完整版 5483页
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+ W9 R( Y, Q1 @ d, [Online ISBN: 9780471143031- O$ F- D6 x& w. c8 g$ Y) F6 p) q Y
DOI: 10.1002/0471143030
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+ u! I4 q( b" c9 w& n. H3 q3 }Table of Contents' C) U, h, T7 k
1. Preface9 \( `) m; x0 h" @0 B& p
2. Foreword) H2 F8 H- d& C4 O+ z5 t
3. Chapter 1 Cell Culture$ W; z! M2 {% t3 V5 m! x3 `
1. Introduction4 l# {0 C' Q* _7 K+ t* L
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
7 \+ N _( |. F3 ~: o3. Unit 1.2 Media for Culture of Mammalian Cells- z! D0 i1 w! r7 B o
4. Unit 1.3 Aseptic Technique for Cell Culture" S6 @- u5 _' ~. k( v
5. Unit 1.4 Sterilization and Filtration
+ C* g( F* |2 d5 l( ?$ ?6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures" T# U3 f2 T, O# q
7. Unit 1.6 Media and Culture of Yeast$ m; Q) H( F- @. \
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
4 G- K* A* ?) |& ^4. Chapter 2 Preparation and Isolation of Cells
, f8 R- c9 B. g' E+ z/ I6 P$ N1. Introduction
) [+ X) T( {) s% ]2. Unit 2.1 Establishment of Fibroblast Cultures
+ Y0 M/ F' o* R' ~2 S# ~% x6 Y3. Unit 2.2 Preparation and Culture of Human Lymphocytes
! z: {; V/ X5 W) B( S4. Unit 2.3 Preparation of Endothelial Cells( Q1 S6 i5 n, I, N5 x! ?
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
' }$ F0 P6 O7 D& m1 g. e5 R. w6. Unit 2.5 Laser Capture Microdissection) }' s4 s$ g& y' h
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures& c/ t5 b0 U# a3 ~& X3 S
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
3 @: f+ Y# I5 r' Z' D( D+ M, A5. Chapter 3 Subcellular Fractionation and Isolation of Organelles9 A% X, k" i! {0 @0 h
1. Introduction
$ Q& t ?" n4 }2 J# S* N4 v2. Introduction
( c3 v) W1 I/ x: R3. Unit 3.1 Overview of Cell Fractionation
8 v2 G; n9 {- r% S" f+ x; i4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains! d. |. @- t3 s. q7 B
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation! n, M0 ?) p4 r6 [7 _
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation1 p! G8 ]& ?4 X* M/ {) b$ B
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient' E, ]$ V8 S m) N
Centrifugation
) l6 m5 |' p" Z- p4 `; k, X8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient# h1 V3 G. h6 o) y$ i) c6 y
Centrifugation* v. H7 R' {! Y7 a/ |
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae! j2 Z3 g; m% x, c6 P2 n
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae2 x1 ? M0 C- H- V
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
# G' W% C( |0 O& Z( D5 F- DCentrifugation
9 I& s1 F+ j* \/ f( V: X" q0 F12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
( F9 B- \9 S+ P S7 M% a13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
0 k% ` z7 i5 _5 G h' @! P14. Unit 3.12 Isolation of Synaptic Vesicles
8 ?: Q3 c* [6 P; M% }4 G15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation" i/ `1 g# U6 t( Q4 |$ c
16. Unit 3.14 Isolation of Melanosomes
]2 Z( J/ @$ J/ _+ B+ q17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation s# G1 j6 R+ {: J# B
18. Unit 3.16 Isolation of Mast Cell Granules
5 |& A" y* o. p& T19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster: x8 f G" S0 s! Q' u8 X
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
2 y& N. H+ H3 Z# f" m7 z/ Y3 N21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
7 o! ~7 h' b5 h5 H& @, c* [8 \22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
8 [% g5 j* f- l% k. l23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes: H) g. ^: N& I# |, H, a
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological1 g% K& B, h1 ^( F
Fluids; A8 ?" e4 _" Q% j
25. Unit 3.23 Isolation of Intermediate Filaments, g: k3 y: L$ Q- x. }
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle5 D) [! [4 J) F! q! `/ J( L4 c
27. Unit 3.25 Isolation of Myelin
5 S! h; C/ x6 q2 c. m2 v3 }: `/ z28. Unit 3.26 Isolation of Renal Brush Borders
. ]7 D! _( G7 S8 a0 q29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
- @" m# ~5 w9 j' Y ?& zFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts- E3 m! n4 [' f0 L+ T' ?7 H* b
30. Unit 3.28 Isolation of Amyloplasts
8 Y2 \; \! @0 H" v S6 E31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
: |' |6 W" e+ \0 g32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic/ S/ a o( ]- `6 L; L0 Q$ v! H7 K- D
Centrifugation
$ C/ x3 P# U D2 M% @2 Z33. Unit 3.31 Isolation of Neuromelanin Granules$ K. {% ?* e( M* _" ^
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
2 K9 _( s; }/ B9 e& [4 j* PDensity Gradient Centrifugation
m/ G. k1 J6 Q* Z9 w# R: u Z. p35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments1 p( e; A! A) R- `! }7 @8 V
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
8 u, J; U, B" |+ \- ?: K9 B37. Unit 3.35 Isolation of Platelet Granules
% I$ @7 j+ q6 `* B1 V9 t38. Unit 3.36 Isolation of Nucleoli( Y {# u4 z S+ L
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins( `( D- a+ s* f: K& D4 |5 u
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates+ L( {2 D+ N7 `& w
41. Unit 3.39 Isolation of Chromaffin Granules
; v5 q* X9 F% T6 N42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
( j/ X* V5 I( Q, s! Q6. Chapter 4 Microscopy
- G3 o: {0 F% s& D1. Introduction
2 [, @$ z+ V9 E) m2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope. I& G$ a$ z8 _4 k
3. Unit 4.2 Fluorescence Microscopy2 T- W: l0 I: S% m& \- c
4. Unit 4.3 Immunofluorescence Staining+ D0 \; l) f! m( p1 n4 |( L
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria* M5 E% q. G# k
6. Unit 4.5 Basic Confocal Microscopy/ g3 _1 j z0 w' t: P
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
' A8 S6 }3 c4 S3 ~' _4 p% g8. Unit 4.7 Cryo-Immunogold Electron Microscopy
7 N: {# L3 @9 @3 j5 j9. Unit 4.8 Correlative Video Light/Electron Microscopy! h: a( y! {7 |
10. Unit 4.9 Polarization Microscopy4 }. E' r8 V9 k5 E& T
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells) J3 `1 c1 F* u$ A9 _# u6 f h
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
! v/ A* e0 R8 I; _# l13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface& ]6 R0 M$ M! }4 X
Events
9 Z, I( c) c! l$ Z3 i5 f14. Unit 4.13 Fluorescent Labeling of Yeast
9 i. w: C; G7 C$ w. \% M15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy' D, a! R. I) ^
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy" X: O/ t M& r& b3 K, W, b1 z5 {/ l
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules9 [( Y3 y, Y e9 N& {$ i! y
in Endocytosis
: Z2 Z. t1 a) z1 p4 p/ I18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology2 q8 N3 b/ v4 \: r
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
* P+ ^% h0 |' v20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
0 b0 @, H' ]6 m7 e) `21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
0 j* ?0 [% J6 C0 W8 ]3 a' }22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
% B( n7 r# R) O3 T23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
2 z( r$ P$ m: r7 d' ?2 A: ~24. Unit 4.23 Interference Reflection Microscopy) {4 ?: \5 K6 {8 a3 {* A
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach8 p S+ {: R2 L
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches( X1 G1 o0 z) g) a3 y0 k
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
: c6 u6 B, J5 b3 b3 S& g7. Chapter 5 Characterization of Cellular Proteins
# @; w0 Q) ^8 J1. Introduction
9 [& M0 Q5 K7 v; U. C) j& r2. Unit 5.1 Overview of the Physical State of Proteins Within Cells ~0 a) L1 g6 \ ^5 x, m
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein% L: A$ N! J X% b
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
- V" _6 r' K y6 t: l5. Unit 5.4 Analysis of the Association of Proteins with Membranes4 d1 j/ @. y- `
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)/ {: _4 V2 Y5 ]6 J. t# h
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass! d# t' C% j7 Y8 s2 b* \
Spectrometry1 j& Y4 b0 C. T, T6 V+ J( Y6 X) i, n& ?8 P
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening, P. |5 w( I3 I& I
Applications
3 L8 [1 {/ R0 k' b$ P* w- D9 k) E; p8. Chapter 6 Electrophoresis and Immunoblotting' T# S% T! v+ u' d
1. Introduction$ ?5 Z8 l) H3 ^! I, I9 U$ s; L& }
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins, R3 L3 K# d# v# T' B
3. Unit 6.2 Immunoblotting and Immunodetection, n2 H7 W. t% q( G' I1 N
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
# G- {" B7 V9 N5. Unit 6.4 Two-Dimensional Gel Electrophoresis
0 Q" K7 p; _. P; L6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions$ V5 F& P( ~5 y8 s7 F
7. Unit 6.6 Staining Proteins in Gels, ~, h# Q- z/ O( m1 ]9 `8 ?
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
; u! ]# q$ j- Q8 K9 H+ r; l9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
% q, s% A" @; m$ u. k$ E10. Unit 6.9 Digital Electrophoresis Analysis1 B! n; r# a1 B1 L/ C$ u( C3 W
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
+ w L$ | |# N4 M12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
7 o1 l) u% z- i6 R2 R# eSingle Cell Gel Electrophoresis (Comet Assay). s: \- e4 {$ R [4 l) @5 S/ {& v
9. Chapter 7 Protein Labeling and Immunoprecipitation
8 e8 ^% r A) ~) r1. Introduction' v" N- y6 x- @5 K
2. Unit 7.1 Metabolic Labeling with Amino Acids
( w9 U4 S9 Z/ F. L6 D5 L# M3. Unit 7.2 Immunoprecipitation$ v) ?) o& `- I' u% X( u- E
4. Unit 7.3 Metabolic Labeling with Sulfate
: }# v1 Y& w! E1 m. b! T; T5. Unit 7.4 Metabolic Labeling with Fatty Acids/ _7 x$ l F; u" V! c1 O9 Y
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups2 R* j1 j5 t7 S
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
3 W5 P8 j2 d/ q9 s7 H1 |8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
1 n" D1 e: _- z* y1 p; c9 L. _' q9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars- J0 b7 W. D# `7 `3 h
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
/ Z( \8 E- Z3 D. h11. Unit 7.10 Radioiodination of Cellular Proteins T K& n3 J5 o' Q3 \' E
10. Chapter 8 Cell Cycle Analysis
6 x1 S# O$ \6 n3 q' W( g) }6 X, q1. Introduction6 @1 a; w0 Z, I+ }
2. Unit 8.1 Overview of the Cell Cycle
. X2 e1 _6 F+ b& J" `3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle* K5 A# [* ^% f4 m2 h7 M& }
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle4 v, y" O: ~& n- z+ y8 ~6 A& [# D4 G
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry. f; M5 |: J, h7 a
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
( }0 t- M( [4 _: n. ]" v7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
+ M0 L6 h0 @. ^) h4 z1 D11. Chapter 9 Cell Adhesion
/ T3 d+ K1 ~, {2 H) {- R1. Introduction
m: X3 }: X2 R! N0 O2. Unit 9.1 Cell-Substrate Adhesion Assays- m* W( O, U2 t& S) T+ M
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
( ^) l* i4 r1 ]8 Z/ ^4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion# x2 J+ j5 _# T' |) V. X& T
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
3 V1 h" \: R C4 D1 H6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
" y0 [# R3 O5 S# c: [# j7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
7 R# O! G) X9 K+ E3 ^# P12. Chapter 10 Extracellular Matrix
6 |$ S v. [. b8 m% G1. Introduction1 q0 b/ M- @ }* P- h5 t4 S
2. Unit 10.1 Overview of Extracellular Matrix+ H+ y2 f8 D8 _9 ?( N1 A
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
; B3 m. W6 G5 V; m/ d4. Unit 10.3 Preparation of Gelled Substrates E2 O9 V2 l; i# X% z: h
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9( ?1 T4 P0 v0 r1 ~3 G% \+ a+ I; D
Endodermal Cells
" t. V9 Y7 E3 _6. Unit 10.5 Purification of Fibronectin
# f9 |# a2 T R# X6 E4 b: `: c7. Unit 10.6 Purification of Vitronectin
& @9 e5 W ^$ t, a9 E! Q8. Unit 10.7 Proteoglycan Isolation and Analysis
$ R/ S' g r u, ~ K0 O9. Unit 10.8 Matrix Metalloproteinases
, [7 ?) Q' o4 ]8 r' \% j! w2 W `10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts, a% B7 |& n; m: M% Q" c
11. Unit 10.10 Purification and Analysis of Thrombospondin-10 a% @! I' C( l7 r5 g6 B; h
12. Unit 10.11 Purification of SPARC/Osteonectin6 X/ g; L! J4 i7 h
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
F/ F- u) _7 Q i5 T* y: K Y' E& D14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly% ]3 Z- `) s! O% [ }) w' G
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
- }6 M) _0 p4 B0 t3 O# l( \8 Y8 o- |Xenografts
4 j. A$ G# z0 z. ^16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
7 r" g$ b6 n/ v3 F: F17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties+ s1 t: g, j0 p z+ B# @
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D1 K2 A4 B) c, W2 a
Cell Migration
' U7 N. K- u3 S2 @+ q1 @19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
. @" t5 p" F, R7 r' x13. Chapter 11 In Vitro Reconstitution
}/ ~3 c) j6 U$ }3 i+ |1. Introduction0 v2 d, G7 I$ y' h# R) I9 ]9 V" q
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
9 C6 c: e6 O' w8 r8 {3. Unit 11.2 In Vitro Translation
% p) H7 g% ~; ^* q. A; V4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
, _6 A& `1 }( J6 O1 d5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes7 T$ ] X/ B% B( A$ y1 Y' D
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication0 v( q! R% |; F
7. Unit 11.6 In Vitro Transcription
! N4 ~: |! K/ k, `* _% g8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells( F3 Q/ u1 f) \
9. Unit 11.8 In Vitro Translation Using HeLa Extract
( o+ G) M4 w& ?' ?10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
! L1 h9 H7 f% H: ^. t2 a/ l7 t& L11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
3 W& i) r7 Z. I3 P E12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts; A7 v6 u6 {+ x: [( w* L! N
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts8 U3 r+ `* h* m
14. Unit 11.13 Mitotic Spindle Assembly In Vitro2 x" q$ S1 h+ H* }; V
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
% N' z- B8 j4 c16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import1 U, Z, d2 `' e) E! \: H h
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
& T+ D: @4 {) B$ Y' U18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
- h* v& H$ {& B/ A. ?19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
+ F7 \3 ^: k7 E5 _* D5 E4 j M20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
$ ^ H3 P9 i# w2 q) [4 F; U14. Chapter 12 Cell Motility9 x% E0 ?$ {% D5 A
1. Introduction. {7 j' @# e3 O$ g
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells* f* }9 Z/ Q8 [
3. Unit 12.2 Invasion Assays
; `! J5 u0 z/ t# s2 P4. Unit 12.3 Cell Traction1 ~0 z+ a8 E \6 D* T* s
5. Unit 12.4 Cell Wound Assays
8 @* T. W6 E* A/ C6. Unit 12.5 Dictyostelium Cell Dynamics
% `' \$ F; k, B$ l' F3 o: Y/ z" o# U0 N7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils. ~! ^6 L, w7 S7 q
8. Unit 12.7 Actin-Based Motility Assay
- D q# x; @0 W9 `3 M1 j* ^9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP4 y# c. ~" R# \' m: l* T# {2 @. D. i5 T
15. Chapter 13 Organelle Motility
$ ?7 S+ v* M2 p2 Q0 E* D1. Introduction$ l8 k; r0 @- l) [4 V* H9 G6 w
2. Unit 13.1 Microtubule/Organelle Motility Assays
9 i9 o! u9 Y8 Z1 J/ T3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
4 U$ {. z5 R2 U: k" g4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
- U: D& \: ?! }' t) n8 Z5. Unit 13.4 Movement of Nuclei
& [0 r3 E+ Y3 s# S& u. p6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching8 F" _( T9 z/ I# r/ W
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly# T) J% K0 s9 f" s
16. Chapter 14 Signal Transduction: Protein Phosphorylation
8 @, W% G. W# X/ C. u# V1. Introduction
/ ~ g4 ~- e) t% v# |) {9 p2. Unit 14.1 Overview of Protein Phosphorylation
\( N) F9 r/ [# G' [8 l4 H2 {6 r) y3. Unit 14.2 Immunological Detection of Phosphorylation6 ~! W" @4 Z/ x. _+ x+ M
4. Unit 14.3 The Detection of MAPK Signaling4 f H" Q7 }& Q2 l
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
: b2 |6 K3 D$ a+ Y* h |) ?+ U6. Unit 14.5 Phosphoamino Acid Analysis
/ {) y! y- ~8 z6 S% q7. Unit 14.6 Determination of Akt/PKB Signaling
9 s$ d: b' ?& j6 l8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
/ j( |$ D# e/ {0 U6 p9. Unit 14.8 Rho GTPase Activation Assays( D/ g1 Y; q; p+ }
10. Unit 14.9 In Vitro GEF and GAP Assays
& G. U& _/ v: S; {- Y" S' j. g/ }8 f11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
; j" O; c5 j9 s# e" ~; B% AEnergy Transfer (FRET)1 D" ^. N+ F7 ~ A9 G
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells0 D8 O% ^9 c6 {0 D
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells% T8 f& v0 W1 H/ t0 w4 ^
17. Chapter 15 Protein Trafficking
8 W& l5 B S# ~8 p1. Introduction
( C' ^4 q: s$ N( L, |/ Y2 u* v$ H$ D2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
# x Z* Z$ P; l4 ?3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
$ Y0 ]( B7 R# \. R/ m- y4. Unit 15.3 Endocytosis: Biochemical Analyses
# z d, w, M/ y" g. U" ]; v8 w! k1 Z5. Unit 15.4 Determining Protein Transport to the Plasma Membrane' m: @0 C# F- Y0 Q! P8 O
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
+ H$ p. \+ ~' ]" Q7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum. P* s+ l1 k& @# Q
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
, x O2 s9 G3 p! M2 ^2 I. a1 V9. Unit 15.8 Analysis of Protein Transport to Lysosomes
% y0 s' h4 |! U10. Unit 15.9 Studies of the Ubiquitin Proteasome System
" U- Y. I0 | R. G; g7 m/ |11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network* z) \4 Q4 I% j2 s* w. y6 w
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
o2 S! c3 D0 R! }13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
; H, e7 A& x2 S, i14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
2 k4 g, d( b6 W# L$ z15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and$ V' S! W# T; R6 h0 R( ^" ?" E* j
EGF Receptor
$ y# F. l3 f9 Z3 P0 Z* y9 z* d16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
3 B/ @9 y+ o/ C- x" H# R18. Chapter 16 Antibodies as Cell Biological Tools, l) G4 V+ F+ T7 V' L" U7 K* l
1. Introduction
: h% M4 x) b# R2. Unit 16.1 Production of Monoclonal Antibodies: n' o' p- v3 m4 C; Z5 {
3. Unit 16.2 Production of Polyclonal Antisera# I; J# {: {1 l6 c
4. Unit 16.3 Purification of Immunoglobulin G, K5 q. Q% s. E4 o* y
5. Unit 16.4 Fragmentation of Immunoglobulin G/ N6 ?. @: `1 C6 D4 P' S
6. Unit 16.5 Antibody Conjugates for Cell Biology
, m7 M$ q( z0 k' m. L4 m! I3 I7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides2 P, S1 W- ?1 r- U+ a
19. Chapter 17 Macromolecular Interactions in Cells, D; ]2 s; I9 R" Z
1. Introduction
5 t7 g; X& d) R' D$ e2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)) j8 r& u: g( @7 n9 J
Microscopy
1 o1 Y# W4 Z% h6 K" r) J& E- u3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
! h# J2 f( s- U4 [5 G$ u4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
1 Z7 _1 q2 v* S+ y9 _( ~5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries8 Z+ q: x( p' k M( M( _& T1 \% t3 Q
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
9 j% H" ^ c$ V% B7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors- z9 f( s9 N+ @; s- Y
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific5 {9 n7 c9 d6 U5 z1 |" E* c
Genomic Sequences In Vivo5 J* m! A6 h4 ~4 u7 m% y
9. Unit 17.8 Isothermal Titration Calorimetry0 F0 U" m0 D7 V; }% }; @( I' y
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells& O6 `8 ~! G Y2 f# S
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking- t! ?1 l# l q/ _( ?0 \: N+ y
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
7 J+ c! z: ]" E5 u' t9 z/ [Interactions (RFAP) in Live Bacterial Cells7 x( a) t' b, [$ w, T
20. Chapter 18 Cellular Aging and Death8 S! D4 ]+ {2 J8 F
1. Introduction
, c1 Y* ]" R7 u# U% A. A) G9 U2. Unit 18.1 Current Concepts in Cell Death
4 i* k' J& J& o9 b) y7 p& W% ^3. Unit 18.2 Analysis of Caspase Activation During Apoptosis; L' ~1 S( A3 K$ O/ Y3 o
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria$ _( D* T0 F0 x/ j3 V; g
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)' D5 W/ [" H5 A! A: ]; b# `$ Z
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death9 D6 ]4 T$ ?# B5 {9 ^$ v
7. Unit 18.6 Analysis of Telomeres and Telomerase
( p- X* G5 ?' z. p8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of/ O, ]& t3 u3 C9 p
Poly(ADP-Ribose) Polymerase
7 r" r$ P7 ]9 n; n9 I' Y9. Unit 18.8 Flow Cytometry of Apoptosis r* p$ p2 I( d0 f: A. z0 ^
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase1 Y3 p5 y* c1 b7 y
Assay
% d; m6 L5 ?2 y( x' y4 c11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis' z0 Y; {) Y3 @. H, G3 v3 Q
21. Chapter 19 Whole Organism and Tissue Analysis4 j0 K9 ~, f; ]) j
1. Introduction
4 X. f1 ?; U8 |! G" \: {" Q8 C2. Unit 19.1 Overview of Metastasis Assays3 G7 U, |8 m+ j ?8 S S) l
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis; D- a# ]3 L7 M! |" @
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene2 z; m( ~) I. X! g( O7 @
Expression After High-Fidelity T7-Based RNA Amplification3 T" g9 ]1 R1 e6 O6 |5 Q
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
9 L6 O+ F( v" I; W1 G* u$ `, c6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model* }3 r# P% U: U- J4 ?' B: H6 B
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo- j2 B0 o2 J# e+ Q ^1 o5 z7 _
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo& {7 Q5 t( T& c3 h% o; G# `
9. Unit 19.8 Embryonic Organ Culture
. }; q! T* x Q5 t2 {: O: {10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin* [: \: H0 g! A7 }7 }3 D$ ], w2 c
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
+ C& S- j4 {9 ]% C' H9 a R12. Unit 19.11 Generation of Transgenic Mice
8 \1 u, y( s W7 K13. Unit 19.12 Overview: Generation of Gene Knockout Mice
1 y% X/ H* K$ |( i1 w8 K; k14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production5 w/ E' H& `* {+ g5 \* V
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection* ~- ?0 y: A' ^5 y# U7 \
22. Chapter 20 Expression and Introduction of Macromolecules into Cells4 d! B0 j) b0 T& [) M% V3 e. A
1. Introduction
! f7 p: J, E* L! l: g; k2. Unit 20.1 Direct Introduction of Molecules into Cells
" o* P. a4 y1 y1 ?2 z2 e# H$ A3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System |1 g" o& G* C5 W$ J! m
4. Unit 20.3 Calcium Phosphate Transfection6 K/ ^7 v, J8 I! D+ I% \6 B# v& f* z
5. Unit 20.4 Transfection Using DEAE-Dextran
+ v# Y' x& J7 \6. Unit 20.5 Transfection by Electroporation' b5 R. {' N) R( A
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents# r) r( }" |+ c4 r* O4 N
8. Unit 20.7 Optimization of Transfection
( u2 @4 h( E# M9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System1 J* J! @% a( m/ k
23. Chapter 21 Fluorescent Protein Technology
" P2 \& z& Q: L7 U1. Introduction
+ G! G% a3 Q5 y" O0 u2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells, ]( F! ~) C. b" M4 m6 }) t9 D% U
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)% z* l" Z: Z$ |: [
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
: i/ i6 ], g# G" K+ |: m0 m& I+ mComplementation (BiFC) Analysis
( h8 f2 F' t. ~" s0 K2 {; s5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
0 `, `9 X" a9 C! b4 P# d6. Unit 21.5 The Fluorescent Protein Color Palette. {" q5 ~4 u3 t5 {
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins5 _9 b% {3 h. L+ T
24. Chapter 22 Cell Biology of Chromosomes and Nuclei( t0 D' w; Y! b {! M
1. Introduction$ c7 L. E6 D3 x: ~! M9 A
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis( y0 Y# P+ k# ^
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples8 }$ f4 G4 ?! E2 D; }# X
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
+ Z# }+ A" g2 _0 ]5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
& [4 q; Z( p3 V/ h6. Unit 22.5 Multi-Color FISH Techniques
% |1 c( {: T- |' e- y, _; u( \# Q7. Unit 22.6 Comparative Genomic Hybridization
$ `' A: w& i4 `+ q; a3 O. G8. Unit 22.7 Sister Chromatid Exchange
, j" x( V& `% o, Z [9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery2 p, } H) \% y& Q# o5 U
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
6 U" \/ U r: f9 Y6 V11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs E' L: F* {+ i# N0 p# s9 y' @- }
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
! T% P& y! w" U: z9 F0 k13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
' c+ K$ P( D& l. C% Q14. Unit 22.13 Monitoring mRNA Export
) Q) l/ K* Q( ^* \, |! G6 X' `5 V15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
( m! |/ s$ ~% y! R* t+ v- zField Gel Electrophoresis
4 f( W. V- Z7 H+ Z0 ~) w25. Chapter 23 Stem Cells
8 w+ a4 M4 e- g6 \3 o( d1. Introduction
( g* U7 v' X2 Y4 g) b1 B2. Unit 23.1 Stem Cells: An Overview
8 h# z4 N6 I6 n0 d5 }( Y( O3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture' V$ F4 r* o+ D. q
and Differentiation as Embryoid Bodies: N m' t- k0 y3 w+ c
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood. ]8 Z; k& V" j: V5 U" n
Vessels
4 }+ z, b" N" |/ x9 }5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into3 s5 ]% Y* A. A1 e* r
Adipocytes
$ A4 L7 ^! N0 O+ x/ \. `, L, g6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
" M2 N- L0 j! D; c7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
( E/ U8 W, E+ \* M6 N2 n8. Unit 23.7 Neural Differentiation of Human ES Cells
" W& o, i _9 j& y4 P2 K26. Chapter 24 Lipids
5 p9 }4 E7 C* Z" ?( o1. Introduction+ I) d) _% p( F% w
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking4 L: b5 v4 N( \: f* `. c
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins: O+ k7 x& q8 {( x* Z% R; i
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
, z, A# x8 T8 {" W5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains0 R( j* v' k! \$ _
27. Chapter 25 Nanotechnology/ ^1 E2 @# x& }* Z+ v4 F8 r
1. Introduction
$ P/ V' \& p) {$ O7 h! O9 i2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes- g& R+ \. R0 o- [. G
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
; e! ]: V8 ?3 l28. Chapter 26 Viruses/ D) G& D7 ~) e* m. [2 W! _
1. Introduction3 H6 T7 a% ^$ M8 W/ O# V
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
$ E3 J" _. g! K5 j3 F' o* D" ?; C3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
4 ?' T. \9 n, c2 L0 N# \Cell Entry+ i- f4 }8 }0 W! E+ q+ t
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
2 P8 ~& _& T) i5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
# n3 c* O& j3 m2 [: \1 x6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
2 e! e8 a# R T" H29. Chapter 26 Lipids
$ H f+ b7 L" _6 u @, V/ Y1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
8 s J/ d' R' B' l1 t# o3 k, fReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication. i* x1 k4 d3 P1 {5 ?
30. Chapter 27 RNA-Based Methods in Cell Biology0 `/ @# y4 i% Y$ A& G# t, S7 N) R
1. Introduction
0 v. w4 F$ a P8 Q) i' O: h1 |2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs3 J- n# T" [9 l; l0 v4 h# y/ [; c
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible4 ~" s3 @. t* U
Rescue in Mammalian Cells
5 a, Y% ]' ]! \1 T6 N4 C& _6 ]31. Appendix 1 Useful Information and Data1 L* ]& _$ x3 ?; J
1. 1A Useful Measurements and Data
4 t9 V7 e9 h& N6 f# k2. 1B Compendium of Drugs Commonly Used in Cell Biology Research& q2 X0 z. E+ r0 e- U5 A3 D
3. 1C Identification of Motifs in Protein Sequences7 t) @ e3 }$ g
4. 1D Safe Use of Radioisotopes
6 g% [! @4 Y; u5. 1E Absorption and Emission Maxima for Common Fluorophores# H3 p/ y9 t5 T3 g
6. 1F Importing Biological Materials
8 T* v2 z5 N7 o- B- T6 ~+ d7. 1G Centrifuges and Rotors& Z1 m P# r' y
8. 1H Internet Basics for Biologists
. O; z6 V" A- ]32. Appendix 2 Laboratory Stock Solutions and Equipment( a1 X* n$ p7 ~! u
1. 2A Common Stock Solutions, Buffers, and Media, m2 s( _8 w! Q5 s; B
2. 2B Medium Formulations
9 O9 ~+ b8 T8 V7 n/ O; W3. 2C Standard Laboratory Equipment
6 [# g5 C" `6 s' e# q7 l5 b33. Appendix 3 Commonly Used Techniques
; H9 f# Z( Z9 w! V& {$ ^* g1. 3A Molecular Biology Techniques
I5 |3 u8 Z% ~/ h2. 3B Spectrophotometric Determination of Protein Concentration
4 i, Y- O1 J/ g' T* o C2 M3. 3C Dialysis and Concentration of Protein Solutions
: n7 v, F- G# V5 K7 W4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
5 L% `' b; R5 l, @! s9 V5. 3E Silanizing Glassware! x! c* N0 `7 q4 ^ Z4 Y
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization6 }" a1 R( f$ P9 B9 G0 M( q
7. 3G Micro RT-PCR- Z) I' b/ z% ~# b" O& |) S/ X) [
8. 3H The Colorimetric Detection and Quantitation of Total Protein
$ V4 @& }+ |! }6 P34. Appendix Suppliers
# u5 e% O1 ~) R( a1. Selected Suppliers of Reagents and Equipment% `4 D! W( \- V( l6 b) Z$ z
9 u, {# F* G) B5 F& i( V& @
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发表于 2011-3-8 15:53
