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: \" M9 U: `8 ]. }8 r* ]' F
4 N& \* w2 M, b! a) E5 @) \$ k, wCurrent Protocols in Cell Biology 2010年完整版 5483页
; q8 F! b6 n9 j8 P( l2 N& a
t: F8 L3 c4 ^0 XOnline ISBN: 9780471143031
6 \" \* C [; b7 c1 i; a# ?DOI: 10.1002/0471143030- d+ a+ _7 _" e* H$ j% u, t
2 s8 f2 H8 J' ^( u o- [# ^
Table of Contents
' X3 U5 H9 @/ h B R3 g1. Preface
" B! K) H# k2 E9 O7 x3 u( r% b: T2. Foreword
" k( H& W B% \/ U3. Chapter 1 Cell Culture. C' q) }0 Z2 k7 G
1. Introduction( i/ U/ j5 ~# k. j) u* z& _
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
4 l; E. y: g' [4 Q; H( l& Z3. Unit 1.2 Media for Culture of Mammalian Cells
/ \$ V, H+ `0 k7 f4. Unit 1.3 Aseptic Technique for Cell Culture
0 ^! t4 t* W. I# G! ?0 B& G. I5. Unit 1.4 Sterilization and Filtration
8 ?2 g% F, A' t: s6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures# r2 h2 b' V" a) e( z% `
7. Unit 1.6 Media and Culture of Yeast
H( D! ` H- k& _! V% ]8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging. H* N+ ]6 O8 N
4. Chapter 2 Preparation and Isolation of Cells
1 v9 i9 m+ a+ ~# n" d- T: c1. Introduction
# f9 T% f. A# S6 H2 y$ L2. Unit 2.1 Establishment of Fibroblast Cultures
$ d; y) O* m2 U1 _( C+ Z1 h3. Unit 2.2 Preparation and Culture of Human Lymphocytes
; _$ h o- c& B7 _" _1 P4. Unit 2.3 Preparation of Endothelial Cells) u1 g" U8 Z+ z [4 X8 W* n
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
5 v. D! x. r! j" M7 q/ l6. Unit 2.5 Laser Capture Microdissection
8 [- D4 ?: b: ^5 Y& U9 n }5 } Z* Q7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures3 _( A3 J, Y6 _4 d2 @/ v. s
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells }: q+ f7 P( ]' v6 Y3 }/ d8 Z
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles& c( K4 T$ l) H$ i7 a8 X
1. Introduction" N, ~8 x: @ t1 h0 B' n7 A8 L" [
2. Introduction, }# q$ h+ m* O/ m
3. Unit 3.1 Overview of Cell Fractionation4 U+ Z# }0 R& X( ^9 B
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains& S2 P, b2 J" ~8 b' n# z
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation# N& g+ ~5 ~' x. B- z# J \
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
: D+ N- `3 M: H# V5 a4 w7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient8 d4 s4 B; l" \) B6 d# j- a
Centrifugation
( y/ C! h+ W2 a8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient5 W' g: a- k( {( F+ C: P* M
Centrifugation k$ \, a: D6 `6 T
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae( K2 n5 R' j/ u5 z1 p1 Z' M0 W
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae/ ]4 w, F+ y" _
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient5 G/ f8 F8 m% E1 Q/ _! W3 p
Centrifugation2 [: X! ]' L+ M* \! A1 G/ L
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues3 c, k1 I2 E! j4 P8 i
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes( [: D+ x, A7 M9 \/ X
14. Unit 3.12 Isolation of Synaptic Vesicles
& M2 X+ N; g/ T( {+ N! ^% T15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
7 O3 X; a* @9 t4 _$ j7 \: B3 K16. Unit 3.14 Isolation of Melanosomes9 o8 o' c- H- K; J5 C+ W
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation- k& m. i7 c: V9 f
18. Unit 3.16 Isolation of Mast Cell Granules
) j0 R7 o! D8 L4 \7 A19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster; j: C& ^! l" V
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas* O! _5 J9 k# P/ p2 F$ b* L" U
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
: k2 V$ y1 C1 x/ B22. Unit 3.20 Isolation of GLUT4 Storage Vesicles/ o- g* c5 U$ d1 N) Q8 f
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
2 v9 v8 R8 T; a24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological6 m4 h6 [0 i9 d5 U ]( [; h7 _: g
Fluids
9 l+ M6 G8 h* y) T4 `; @25. Unit 3.23 Isolation of Intermediate Filaments) k& Q" `& E0 N- `$ L
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle) ^6 }& c" k" U/ A% B
27. Unit 3.25 Isolation of Myelin# I N$ B4 k! D4 h m" W- P
28. Unit 3.26 Isolation of Renal Brush Borders
; S4 `: O' K) t; l$ V29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane8 U I: G, V' I$ ]
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts( T5 K; j3 `( ^% L/ g/ x
30. Unit 3.28 Isolation of Amyloplasts
7 \; T. l) w3 v31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins# D, p- U) L4 E1 v" q4 a
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
: c8 Q, t5 Z+ o* UCentrifugation
/ |& s% _ G. T8 h2 U0 a33. Unit 3.31 Isolation of Neuromelanin Granules
5 Q7 Q, k* C2 ?+ S. [9 H3 a34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and, V8 E2 Z x1 W8 r: W
Density Gradient Centrifugation3 }" N1 }( L( o; {8 i
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments7 e: k. E- T1 i. g& v& c
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation, x8 \% D* i% }0 _( P! W8 p& ~+ h' s
37. Unit 3.35 Isolation of Platelet Granules9 y8 t$ e3 d7 X, A9 q
38. Unit 3.36 Isolation of Nucleoli
2 @* n: [- r5 L/ n! n5 a39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
' B8 t, d5 ~( Z# X; d40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
8 \7 V, B$ B9 c8 j; d' A' ^41. Unit 3.39 Isolation of Chromaffin Granules
* k+ W1 w3 R" I9 g2 a1 R42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
$ |% n" _$ Z! V8 k9 P6. Chapter 4 Microscopy g+ l3 a: K+ _+ o" p. V/ O8 Z$ F
1. Introduction
, H9 N$ Z. S9 f0 G' q1 [; _2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
6 x3 C2 a5 L2 J. ^8 O& u4 b3. Unit 4.2 Fluorescence Microscopy
- F/ K" I8 u8 g7 U( b4. Unit 4.3 Immunofluorescence Staining
5 A |1 E; R- }5 H0 a5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
$ f& u) ]' l# L6 t. f3 B6. Unit 4.5 Basic Confocal Microscopy, W2 e1 G( E) R0 W1 N) O% L
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues. C7 g9 h! e- _' w
8. Unit 4.7 Cryo-Immunogold Electron Microscopy
7 r0 r8 e4 K. c- U7 c5 g; [/ q: T6 l1 k9. Unit 4.8 Correlative Video Light/Electron Microscopy
% T' j/ M5 U( o1 f* a) F/ d10. Unit 4.9 Polarization Microscopy: [0 [4 D- q8 n8 H$ f# o5 v/ T
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
- V4 m. F' |$ k9 s% u1 v12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
) t; ?- h! Y* {$ _$ g1 T13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
b+ O8 i. y, G" i3 dEvents! i" X8 i' v# t5 {& |" K/ m. s a# }
14. Unit 4.13 Fluorescent Labeling of Yeast
" F; e/ S9 A# Q15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy' _5 ~# D; q) C0 f O
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
# ^- q4 H9 {) t: E7 s$ E O1 Y17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
7 ~9 p1 A. W8 Zin Endocytosis( X0 \% j8 H' e+ C0 }
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
6 u @9 t% E8 C19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development" v! r M H! E [
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
3 ]( F# f% w7 h# ]) T# ~21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions. H1 d; x; n. y+ Y
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
/ _. p$ l. z( J u( n23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics4 \& _4 C/ P. O* P
24. Unit 4.23 Interference Reflection Microscopy/ b% V6 A( p- B" P0 w
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
* ?) x6 B/ b8 x4 J/ O0 O2 h26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
x+ }) Z6 J% E1 H27. Unit 4A Organelle Atlas: Appendix to Chapter 4
3 R: B- t8 k# l7 ~2 l; J1 r7. Chapter 5 Characterization of Cellular Proteins: P/ I- q7 Q% R( l q! n' v
1. Introduction
" T0 d# |/ l( L2. Unit 5.1 Overview of the Physical State of Proteins Within Cells* f$ A4 t5 }" I& q
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein( i- t4 Z* l( x- ]$ c3 s
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients/ l. r2 x, E) l" e! `
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
( `& O. q. I9 h3 w2 x/ N6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration), c' X2 o; m4 j0 I1 @
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass7 i7 s6 V: y4 E- D7 ~4 W: a
Spectrometry
; t/ r1 L# s0 i9 b9 y5 S4 s/ k8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening# Q5 a5 N; O! W6 x- u' E
Applications
6 F! J: f- p t4 x' _8. Chapter 6 Electrophoresis and Immunoblotting
+ ]* l7 `% b% H- m- J6 r1. Introduction
. C' D( B) `/ M/ Y2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
+ v& g$ t' M! X2 i& N/ G4 a3. Unit 6.2 Immunoblotting and Immunodetection2 g9 R5 g0 T, W
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots# ?* q# z+ V/ G2 r0 ?+ U
5. Unit 6.4 Two-Dimensional Gel Electrophoresis) j* `$ W1 Z/ q2 u; C
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
) c2 N; o, s4 Y- E- ?+ H2 f4 H7. Unit 6.6 Staining Proteins in Gels
) \8 v, L" ^5 N% u( v+ z T& z8. Unit 6.7 Agarose Gel Electrophoresis of Proteins6 c1 }# E$ o6 X+ R$ ~ o4 ?- X
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots( G( F9 ^; n6 k ~9 C- L
10. Unit 6.9 Digital Electrophoresis Analysis# l, ?! {8 I) j1 @+ L
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
7 g& u8 R! o- s2 S: d$ ?+ m* q/ {12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of W+ n) D! r2 j7 y: P2 h
Single Cell Gel Electrophoresis (Comet Assay). a/ a- W7 E3 D# D( S
9. Chapter 7 Protein Labeling and Immunoprecipitation
( Y, U2 x- ?/ n1. Introduction$ _: n7 R! v, X: I1 @ j& \6 D
2. Unit 7.1 Metabolic Labeling with Amino Acids
- b6 I3 B1 ]0 D2 g, N/ p3 q3. Unit 7.2 Immunoprecipitation0 @3 C8 K) ]/ h. t7 y3 A d. H
4. Unit 7.3 Metabolic Labeling with Sulfate
. A3 E; R+ o9 ^7 l7 d$ B- k! E; A+ _4 A) w5. Unit 7.4 Metabolic Labeling with Fatty Acids$ _* j+ B9 K$ H# h5 C; _
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups, Z# j c ]( v8 y
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
3 s2 [4 w, W6 t6 t1 I, p @8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
; w- i" d% B. T7 N K# Y9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
2 f4 ?$ W* i* |# d) y7 a& \9 Y10. Unit 7.9 Analysis of Oxidative Modification of Proteins
9 `" g4 K, h: C0 N, Y9 N* |9 h11. Unit 7.10 Radioiodination of Cellular Proteins# ?; D; y# X, a4 Z/ e
10. Chapter 8 Cell Cycle Analysis
1 U8 }9 a1 W( Y% L& o f- J6 c1. Introduction1 F- X, @+ a2 J
2. Unit 8.1 Overview of the Cell Cycle
! K8 W) H( h" Y9 O3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle0 l" v/ x2 f# U0 ^, P: |
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle# W! j w# k' ^- [8 O. _
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
4 t6 l9 x+ Z( j$ o# e X2 d7 h6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
M6 C9 ]+ M4 U' J3 v7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry. b6 c1 K n7 |. w
11. Chapter 9 Cell Adhesion
) o W3 A5 j* o- ?. z. a1. Introduction* h r2 A/ A* G; i0 s
2. Unit 9.1 Cell-Substrate Adhesion Assays. E8 M, e, e; N& | ^+ X& X
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force" C% G& @6 \! D2 F" N
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion6 F p, n8 y/ F. d9 v9 Y; |5 }0 Y r
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion! Q3 m9 q3 K* D* m8 }
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
2 e- f8 D0 P t2 {( k. V9 b7. Unit 9.6 Measurement of Adhesion Under Flow Conditions R A/ l- c6 _: E$ ?% r. A
12. Chapter 10 Extracellular Matrix
5 S% f" P# R+ }9 W1 T1. Introduction
; m4 _+ d. F* Z3 r) e2. Unit 10.1 Overview of Extracellular Matrix
9 V. [! B6 C) r3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors! T9 F7 T3 E4 J# y. p' c; ?
4. Unit 10.3 Preparation of Gelled Substrates9 p1 A" A9 p2 q$ \6 |" P
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
# X* E; |; N$ f5 D2 T5 eEndodermal Cells6 h( V: `( [0 P# U
6. Unit 10.5 Purification of Fibronectin/ P. o, w8 L. m8 J$ b4 g3 Y( h8 \
7. Unit 10.6 Purification of Vitronectin& P7 d! o2 N3 [% Q* S* d: }7 K- ^% ^
8. Unit 10.7 Proteoglycan Isolation and Analysis
6 [( j* L4 p$ w0 k% @9. Unit 10.8 Matrix Metalloproteinases
; O6 k i$ v9 u; i10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
9 H( v8 s7 G. t8 A- g/ D11. Unit 10.10 Purification and Analysis of Thrombospondin-1
# z& B) Q- B" F9 k9 h0 C# B6 l- I! a' f12. Unit 10.11 Purification of SPARC/Osteonectin
7 c+ g% b V* Y. d+ b, i; J13. Unit 10.12 Analysis of Fibronectin Matrix Assembly. ~' {6 `2 d0 a5 \* C0 d
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
+ q0 O i b/ f- J4 [* I4 }15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor/ q: Y4 W% L! D
Xenografts3 _" O! P" M' X: c8 `" y
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
$ q& x) x, u8 ?* K17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
- {8 n7 u6 |8 [* i4 D18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D7 f, R) Y( H' H% n
Cell Migration+ t, G. B/ v2 c- ]4 T0 |2 i5 |" v
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
; ^3 z- h* L$ N( u1 `; j# m13. Chapter 11 In Vitro Reconstitution: E' M3 y+ [) s
1. Introduction; |# I9 U) M) j( H( p. ]
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
; {9 O. G& p# e6 w3. Unit 11.2 In Vitro Translation5 r/ ]2 a! N! A9 g# ^
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
& T, P+ v% O* Q$ f. C5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes3 [$ j& j2 p& b* h7 K) k3 Q' R
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication5 P$ a: W9 }3 p- J
7. Unit 11.6 In Vitro Transcription
$ l, T/ E, C) {, c8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells' S4 G3 x; r; v x" c
9. Unit 11.8 In Vitro Translation Using HeLa Extract/ c! `0 }6 H5 A: v8 ^
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
; p, n6 I8 v/ _' F" K8 U11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts \: U% z3 K9 V5 H9 G3 B) v$ Y+ h
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts3 M+ H6 ?) y H0 E* |; @' r
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts* @/ [' `0 x/ R$ K
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
* W+ k9 `& J, z15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
# x! J! g' \% }1 J$ }16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
* r {( U1 }" F7 c6 \4 C- F0 [17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import1 D1 W w+ Y+ \; x3 }8 K1 P
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts4 T v/ {. t7 }" J) p
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
0 ~% T# X8 T4 j+ d4 u20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
% y& u9 R% Q- B3 ^5 I, h* s8 B, C- V14. Chapter 12 Cell Motility) B) M) E2 @& ?3 B) T7 {" T
1. Introduction
2 k5 G' }0 O% ^3 g; Z S2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
5 [( D v" }- a% E# ^3. Unit 12.2 Invasion Assays5 ]" ] n2 X2 }4 r: K
4. Unit 12.3 Cell Traction
% G- W' T+ q' }1 x0 m5. Unit 12.4 Cell Wound Assays
; F, i' `4 X. F8 h$ w! L7 z! |6. Unit 12.5 Dictyostelium Cell Dynamics0 a* `9 s0 {1 A5 f% w" {
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils j- P/ W' n5 A! b/ C" A
8. Unit 12.7 Actin-Based Motility Assay! V8 g, d$ f5 x/ l" y4 O
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP) O, V9 v! Z# |% M j/ j7 H' z' [9 B
15. Chapter 13 Organelle Motility
o4 Z) z' x) z1. Introduction
( {# q, ]- P! P" p2. Unit 13.1 Microtubule/Organelle Motility Assays
@# l- D2 y$ Y0 L3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
5 B5 y- v2 e. c+ s0 j1 i7 v4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
9 q C" R* b1 g' Y5. Unit 13.4 Movement of Nuclei
4 l, Y8 J4 d. x6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching3 J5 z( F* @6 J9 }9 T( ^% p/ f
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly; b( f, _) K3 @! r
16. Chapter 14 Signal Transduction: Protein Phosphorylation2 K% W$ S! M% c/ }
1. Introduction
0 D2 b. t3 F+ q" p2. Unit 14.1 Overview of Protein Phosphorylation
4 u7 h5 l! v4 D) E) F5 s, V3. Unit 14.2 Immunological Detection of Phosphorylation5 v; k. m$ B, o# v* Z
4. Unit 14.3 The Detection of MAPK Signaling
8 e7 E1 u6 H6 t5 q* A3 S5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
" w! G. x( ] }0 A; I6. Unit 14.5 Phosphoamino Acid Analysis
) `$ V4 f+ X4 Y! V, w7. Unit 14.6 Determination of Akt/PKB Signaling
6 S8 g2 u5 A5 ~6 y8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
( Q+ l! ]" x2 O2 r0 |* i9. Unit 14.8 Rho GTPase Activation Assays6 b) y8 X7 D) P. ^
10. Unit 14.9 In Vitro GEF and GAP Assays' M& r6 Z5 j) V7 S3 u8 i
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
/ }; }1 Y# H5 q) vEnergy Transfer (FRET)8 B! h7 L$ Y0 z( e3 {# }( ^2 x
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells2 {0 y* c( @4 o# h* q
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells9 t+ ~& W* D1 } ~
17. Chapter 15 Protein Trafficking0 \- K, I& i& S# Z f% [
1. Introduction" E' U& b$ V8 Q9 \7 i9 }
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways" M+ ?+ J. I1 C! c" o: r
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
7 B6 y/ \: L, V. g5 {4. Unit 15.3 Endocytosis: Biochemical Analyses$ Y% Z. _7 H. v8 K3 @9 M
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane. u* t, B8 B: g h% ~
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells0 y. L% A N, v3 z, _
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
% \! I( X1 ]( ^- r. x* m+ |5 K2 P4 V8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation e6 H3 R7 K2 r8 B5 m. R
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
0 a+ Z! d) f9 r4 ]3 b' A10. Unit 15.9 Studies of the Ubiquitin Proteasome System1 U m# ?+ h& Q. G9 J! l k6 ]. ]
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network9 e) m4 @! F8 I, R* }
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules0 ]8 ]& G7 |5 ` R4 a) D; q
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
4 N0 @: v* ?0 H2 m. l" b/ P/ K5 Y, C14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
. u$ F; z, |, C* h9 ?2 O15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
0 y& [6 C8 O- p( a6 C3 c! `+ h0 |( ]EGF Receptor
& L& v) u% o9 I5 |2 W( v, I16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
( a0 C# T* ?5 A/ C$ P18. Chapter 16 Antibodies as Cell Biological Tools4 T4 V2 x2 L& b, S/ g
1. Introduction
- a/ C. [! e( A# r& A2 U! W2. Unit 16.1 Production of Monoclonal Antibodies, A: Y9 W: v. C
3. Unit 16.2 Production of Polyclonal Antisera: {& G9 b1 E% h/ ]
4. Unit 16.3 Purification of Immunoglobulin G) T" q7 a3 {4 n1 e; o1 Y0 H$ B
5. Unit 16.4 Fragmentation of Immunoglobulin G( a: \$ c1 l: N) c+ G5 P) w6 o: |
6. Unit 16.5 Antibody Conjugates for Cell Biology
/ ?) s; a: j" \) A5 R2 t% m; j7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides3 o/ `- L- _2 R' }3 R' K4 \
19. Chapter 17 Macromolecular Interactions in Cells
) h" {; ^' F8 {3 _5 q1. Introduction
6 \; [ i# @, E* }2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
9 G) B1 V# ~" l. W# D/ iMicroscopy0 `# `, h T2 Y* u$ ^/ L1 ~& C
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis5 W) S9 j: v9 f8 j
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
$ L7 o- |8 G, F9 I5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
7 e5 X+ ^) U! m' p4 n4 p% q6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
. V0 [* B% m$ n: z, C3 o1 V) u( t7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
$ a6 \7 K+ c: \8 j* m8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific _0 n$ G3 K0 U: S" r0 ^
Genomic Sequences In Vivo
' M# W+ f/ @8 v% ^2 k0 S; [9. Unit 17.8 Isothermal Titration Calorimetry
; I4 p. {1 x6 |/ o5 s7 s10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
, G D5 Z# f3 G5 n11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking" u0 Q. \# y' P/ p% o1 ]
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
& @! j. e$ Z) A+ MInteractions (RFAP) in Live Bacterial Cells
- l: ?0 v: `1 F& C9 X1 R# ~20. Chapter 18 Cellular Aging and Death
$ c! I* F( Q& O! t, h. C' a# A6 K& e1. Introduction' h& H }, k. z1 N: f* y( w8 `
2. Unit 18.1 Current Concepts in Cell Death
% B& v* e9 X0 r* A o3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
1 B- G% u( t5 J7 k, P4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria' C* E, V0 |, t- {4 s. R
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
' W; A1 R. {8 B6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death2 N) S9 e! K! q
7. Unit 18.6 Analysis of Telomeres and Telomerase
4 a+ i' i& k/ T, z/ Y* y2 m' M8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of$ {- ?% x4 {9 V
Poly(ADP-Ribose) Polymerase4 R* C- G) Y4 x3 Q t }% E
9. Unit 18.8 Flow Cytometry of Apoptosis" w8 R1 U' W3 A2 t$ h
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
/ I& [4 W$ O/ O* sAssay) g4 a+ o' P, Z/ L1 @
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis. [2 t6 E+ z! T7 R! I. a# g
21. Chapter 19 Whole Organism and Tissue Analysis
3 `+ A! S& @/ X: Y) [- l1. Introduction
# ^+ @0 H( x8 \8 M/ e2. Unit 19.1 Overview of Metastasis Assays
0 r' J9 [9 r4 p# h. _, ^8 m; P3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
3 K* U) T2 N' f" e! j+ x4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
+ l/ I8 v% z( G. a+ u0 @Expression After High-Fidelity T7-Based RNA Amplification# S3 I8 J) u0 b# r
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
5 b" U+ E# T8 l9 G* l6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model3 e/ z. T6 A( R7 ~
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo: S1 _1 k1 P. U. {. U
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo" C, D: S* m" |: _9 u8 s; P
9. Unit 19.8 Embryonic Organ Culture. J4 h, R( l' w# P% W
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin; ^- `' S/ X7 O& N7 n ?
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice: l, g, L8 n' H2 l# f
12. Unit 19.11 Generation of Transgenic Mice
1 C8 m. J$ d/ d G0 i13. Unit 19.12 Overview: Generation of Gene Knockout Mice
! [% k2 p0 d; R" [7 h9 p2 [2 e14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
- M* P u6 ^" p+ ]7 C; E15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection% H* P y0 u, V [- x
22. Chapter 20 Expression and Introduction of Macromolecules into Cells+ G* s0 G8 Y) \1 D
1. Introduction* F9 ~4 @: e4 a d* N% U$ k
2. Unit 20.1 Direct Introduction of Molecules into Cells( G4 A( Q& v/ A2 Z2 M
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System1 \! i% ]8 C5 z! c; o! F
4. Unit 20.3 Calcium Phosphate Transfection
1 E1 A0 ?+ M" H# G. [$ q: y/ |5. Unit 20.4 Transfection Using DEAE-Dextran# J J+ B1 w- \+ K( D6 A
6. Unit 20.5 Transfection by Electroporation
; _2 z2 g3 V \4 i/ N7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents5 t: J0 H" T+ T7 ]
8. Unit 20.7 Optimization of Transfection, f& x. J9 W2 {. a+ ^7 E+ ]
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
) w; t5 k/ B3 |) R23. Chapter 21 Fluorescent Protein Technology; i& h/ l9 o& T' y } o+ T9 T B- y
1. Introduction! Y- b9 w0 _' i$ O3 ?4 W6 S7 M3 Q
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells9 Z9 Q/ K/ q& d
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
$ p" N) k8 W' B0 R& [3 r2 n4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
$ E5 D2 z/ l2 M" X0 h1 ^2 AComplementation (BiFC) Analysis" L; m; f- @, ^: y' S% F
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology( Q# ?$ q$ n' b n3 J
6. Unit 21.5 The Fluorescent Protein Color Palette
+ I8 c F5 c! n7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
5 ] L, c* r% F# j24. Chapter 22 Cell Biology of Chromosomes and Nuclei
/ E9 a9 [4 @, @3 ]% E1. Introduction
1 Z% H0 R0 o8 A- W; n8 B5 p2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis; \( v3 \1 \1 l
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
9 Q& L# _+ h$ g) p% P$ K! k4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis ? }) G! X. C% l' [2 j
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
: q6 k. t2 `; f- [6. Unit 22.5 Multi-Color FISH Techniques5 `' T* k; J! a5 @5 k' U9 U
7. Unit 22.6 Comparative Genomic Hybridization
9 e9 w% M( B! N! u5 I6 b( p u- q8. Unit 22.7 Sister Chromatid Exchange
3 \; n- B* D- l0 [/ A9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
; \7 @3 V1 H9 `, I2 u" r. J10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes9 S- C8 v/ p: ?$ V4 g
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
( z/ f; Z5 @& e12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
; `! F% l9 v; ]- q13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
w+ u3 i% n* {3 C: S# ]14. Unit 22.13 Monitoring mRNA Export# C3 r$ Q) h: t4 ?2 E5 k
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
6 v V3 S% _' RField Gel Electrophoresis9 C% H/ b' p& t7 n% a
25. Chapter 23 Stem Cells
# Q* w! U5 X, [# C+ y1 O4 K, s1. Introduction2 K3 U5 V! k7 D+ }9 ]- [" _. P
2. Unit 23.1 Stem Cells: An Overview7 W, z+ o: `- w' t6 k* @- r( j
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture6 `( r( s' ~& M
and Differentiation as Embryoid Bodies5 h& |9 x }& k( G
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood7 w4 ^& x9 S+ K* s, g Z
Vessels8 k4 o3 ~% v; A1 O5 p1 t
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into, n$ Q2 N. H9 ^$ h' l [; p
Adipocytes
8 d9 M+ f" L0 A. e6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
6 q# ~, G, _/ j8 @7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells: \: g3 B$ ~* K" A) w# e
8. Unit 23.7 Neural Differentiation of Human ES Cells+ d# ~2 t! x n
26. Chapter 24 Lipids
& c! k6 v4 V, \2 @4 ~( i1. Introduction1 f- q+ z9 w) p0 P; r
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
# T# s y$ A# F/ a3 B3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
1 q6 J. m5 n6 x. Z: q2 P6 R, l4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film/ @6 |7 W6 _2 Z4 Q; J0 S+ v/ S
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
! f/ ?& L# z# F3 s27. Chapter 25 Nanotechnology
; K9 y. e8 O: p6 ^1. Introduction
, r. Z- x- z- E2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes( u* J$ F- m+ W; y( ?
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
& {9 b' J+ D3 R6 r28. Chapter 26 Viruses/ V/ K& k: D' h# {1 F; f" l
1. Introduction9 Z+ z" e! e3 P) Z3 `
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
. L ^0 l4 Z: h, `3 E# D# v$ U3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
* u8 E0 h8 A, D' `0 mCell Entry# }% P5 r6 Q! T% c% x
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection+ ^5 Q3 w* {% ?/ e& n. \ T
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
8 u9 p( w! M* \* y S0 C6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission: w7 X4 W' w0 m+ U( g
29. Chapter 26 Lipids; u5 u- }5 c6 f8 C, ^
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by; g1 D% r* J& R2 O9 ~
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication( W7 j& U7 H! p+ M( l8 b4 p( f
30. Chapter 27 RNA-Based Methods in Cell Biology8 ]' ]8 e% f M( M
1. Introduction
, t& F* E- L# S0 P/ o2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs b& y4 v5 ?- x
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
: H: D; u4 Q9 l1 y# S, JRescue in Mammalian Cells1 C# C F/ I% i: A+ W
31. Appendix 1 Useful Information and Data! `) m( k2 B1 \' L$ I4 A' T
1. 1A Useful Measurements and Data
1 r! J8 y# @! `$ U5 b, @' M2. 1B Compendium of Drugs Commonly Used in Cell Biology Research' u' i! d5 V$ q" B! j# _8 V j
3. 1C Identification of Motifs in Protein Sequences5 B" e) H0 Y9 Y5 o
4. 1D Safe Use of Radioisotopes
& `: Z+ J" }# m" i5. 1E Absorption and Emission Maxima for Common Fluorophores
3 _' Q4 |6 n# K% @6. 1F Importing Biological Materials
! D* M8 j) C/ v( w; X6 ^7. 1G Centrifuges and Rotors
- L/ a4 E2 a w, x9 a8. 1H Internet Basics for Biologists, Q* ^/ Q, l" R+ n* I
32. Appendix 2 Laboratory Stock Solutions and Equipment$ K. _8 a8 i9 T; J) T* f* a
1. 2A Common Stock Solutions, Buffers, and Media
5 r2 @8 V7 O/ ?' r2. 2B Medium Formulations
& h8 e, e( x; v. Q' j. f# Z: f3. 2C Standard Laboratory Equipment9 z- C& p! J1 h' m
33. Appendix 3 Commonly Used Techniques$ i! W* C9 g0 ^( f8 q* D
1. 3A Molecular Biology Techniques
1 N! U0 u5 s' k( Z# I5 t4 n: _$ n2. 3B Spectrophotometric Determination of Protein Concentration7 h9 \7 f% |3 a/ q# ], M
3. 3C Dialysis and Concentration of Protein Solutions
9 ], X4 b! ?# x$ N o' s4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
4 K& c8 v& z$ J) j1 g5 x5. 3E Silanizing Glassware
. M2 m2 J3 r5 b1 r: n3 z5 S6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization! A% Y( Z) \# [; X
7. 3G Micro RT-PCR
! C: Q8 w4 ]# x$ ]4 o/ \0 t8. 3H The Colorimetric Detection and Quantitation of Total Protein( N3 ?2 l5 R4 s3 u# y6 S& m/ ~
34. Appendix Suppliers t, R6 J$ W: D3 d7 J' t
1. Selected Suppliers of Reagents and Equipment
& `0 `0 ?) G% s& Q5 r2 l
5 g* a; [! T0 ^. l8 r |
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发表于 2011-3-8 15:53
