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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 9 f7 c" G+ l2 d7 e4 L8 t
* t1 l* R: W/ ~9 f5 B. F$ CCurrent Protocols in Cell Biology 2010年完整版 5483页7 I- O/ c' k {) ?0 y
) c; _: \: h3 v+ @9 [ n) M
Online ISBN: 9780471143031
, f, Q& J& y0 z& e, l$ M. o, ]DOI: 10.1002/0471143030: [0 g% t; Z: R/ Y- p5 d
/ B" l) D" F/ p+ D, ^' o
Table of Contents
9 j* Y" ` _; @, r8 x% h1. Preface) j b* U: r' e/ ]; h( D3 I: Z
2. Foreword
8 i X' ], @( ]& R" j* Y5 e3. Chapter 1 Cell Culture
8 }: \5 D. V3 `# W/ R% X' \1. Introduction# I+ D) k7 H r
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture; [0 C% `# X$ [0 N, u
3. Unit 1.2 Media for Culture of Mammalian Cells
( H* @3 X7 o9 U! ^3 [4. Unit 1.3 Aseptic Technique for Cell Culture
! G1 E: n/ l* {, G3 q9 y5. Unit 1.4 Sterilization and Filtration
) O& z9 @- _# u u6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
7 u2 S; |; r+ _& c& y7 m3 p7. Unit 1.6 Media and Culture of Yeast
y; l: K9 |* g, [9 C) D2 p8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging* t3 J: G4 D$ j. a
4. Chapter 2 Preparation and Isolation of Cells
2 l9 x4 ~+ z# [1 ~' I1. Introduction
, i3 i3 } n% N3 X, f p8 E* D2. Unit 2.1 Establishment of Fibroblast Cultures
; u& m/ W- O0 h7 }! C0 C+ V" q5 G0 B3. Unit 2.2 Preparation and Culture of Human Lymphocytes4 p" h/ D. V- R4 D$ _5 F6 a" X
4. Unit 2.3 Preparation of Endothelial Cells2 O- c/ H$ J# K, L! x
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation2 F' c; K2 t' I( J& P6 s* D ^1 q5 G4 d
6. Unit 2.5 Laser Capture Microdissection
0 z% j3 L. b9 B4 _- \7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
/ T( }) O* X' Q" J2 \4 d2 f8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells& }; p; Q! N9 r
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles2 Z2 k0 C# h- L2 E
1. Introduction+ E/ n' b9 n, L8 G: w* o, q w
2. Introduction
, Q; H2 m0 m4 \ ~9 i+ U3. Unit 3.1 Overview of Cell Fractionation* L; N0 E t( W R: E; a( V
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains1 Z. @6 ]$ w. u: @7 z' E
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation U K: [0 W- y5 {, n
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation# H- y* b, j7 Z1 q, Z: Q
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient. M0 `$ A( X, R$ U0 r
Centrifugation
( Y- j% N( h c" X# i- F4 ?8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
& \' a9 A1 C T5 r( D; H) YCentrifugation2 z4 w9 B' P0 ]/ u; [
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
4 U7 v4 X4 v2 S10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
7 K3 q/ ^' I7 |, C, g S11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
' t" [4 M0 I) Q8 `* kCentrifugation
; ^* i. q' w( n8 W12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues( P& R5 Z; K( M0 [5 x+ R {
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes7 ~& m1 R: Z3 E/ L7 _
14. Unit 3.12 Isolation of Synaptic Vesicles
; y; R6 m% m8 O0 x/ M15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation8 f" U2 l, ?" j; o7 k O" g, c8 H
16. Unit 3.14 Isolation of Melanosomes1 E8 ^1 v- b' v* d8 @
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation. l! G z' c% e6 y5 j0 u7 X
18. Unit 3.16 Isolation of Mast Cell Granules
, f; h6 W+ d" q' }! v19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster; X) ?% l7 \9 H8 R- X1 @) H
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas2 ~! Y! s u+ d4 g& W+ `$ T: I
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons7 T8 ]$ r3 Y2 [( g1 e
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles5 `( `# @4 S+ }$ r# x
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes" O$ W' w+ n, ~/ f
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
9 ?; v/ T+ A$ H% T0 W8 fFluids5 p2 f; C# C8 a# s" r6 y
25. Unit 3.23 Isolation of Intermediate Filaments, `% {3 y. b( L' T( m2 l; p
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle$ r+ h" B; ~% g! l
27. Unit 3.25 Isolation of Myelin
$ J7 @5 y( _+ i3 w& P- ?: U28. Unit 3.26 Isolation of Renal Brush Borders
) E7 W5 H) F+ b29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane0 u0 V4 f* Q$ \) G2 V
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
, [5 j3 R# o; s5 ~0 o7 p# ]30. Unit 3.28 Isolation of Amyloplasts
' b; p/ E7 c5 G% l4 V+ K31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
' Q$ C; @3 L* i; B `- z* j e9 e1 Z. U32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
/ c o- [6 M" h% k& E B4 ?Centrifugation6 c6 ?0 o/ |& K; Q f6 F
33. Unit 3.31 Isolation of Neuromelanin Granules7 j9 N- A [2 O, P: L1 E
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
. G, E, C+ w1 I5 Q6 N! nDensity Gradient Centrifugation6 x* \2 _: T3 o8 h) J* N
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments& M+ U5 \4 W* e5 E. n
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
) }+ A) ?7 T, I/ _+ T37. Unit 3.35 Isolation of Platelet Granules' j: z+ ]0 ^1 F5 @2 ?7 |
38. Unit 3.36 Isolation of Nucleoli" d5 m8 J$ z& J7 G9 W) s& B
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
7 X0 w! H7 [( i3 v ~40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates( N4 s. u2 b2 Y( ^
41. Unit 3.39 Isolation of Chromaffin Granules% x& L1 {7 Z' j5 M
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines4 \6 R$ O' X5 J( |6 }+ x# a7 @
6. Chapter 4 Microscopy
: E- J. D, ?6 P1. Introduction% [* u, ~4 Q6 F# a+ V& |8 _
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
8 y6 ~7 p! n% ^8 x& Y6 c) h0 F/ f3. Unit 4.2 Fluorescence Microscopy' f* Q9 L4 a* ] [2 w# ]# H
4. Unit 4.3 Immunofluorescence Staining
% V2 @! B3 ?3 \, n' i5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
# }# {* |6 L7 V4 l/ q2 V9 \6. Unit 4.5 Basic Confocal Microscopy
1 _- ~7 e, }( p$ X+ J7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
; ?3 ?. a q& F/ O% h8. Unit 4.7 Cryo-Immunogold Electron Microscopy6 w- t. F4 o# t
9. Unit 4.8 Correlative Video Light/Electron Microscopy
7 X. h8 ?+ \( i' H1 F10. Unit 4.9 Polarization Microscopy
) V3 _0 j; f) X/ F/ L; c11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
+ b8 V7 t8 ~( M12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues, z# Z( A2 }* P9 F( F" B7 Y* a
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface2 x% a9 L$ X3 C. D
Events
, `# M# v3 i6 T2 A14. Unit 4.13 Fluorescent Labeling of Yeast2 z5 b5 a8 [+ E4 P- d0 v' A4 T
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy# n6 n" h" I; B2 m: N
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy7 [: g4 Z, ~( b. J
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules& q+ W& f: M- M
in Endocytosis: a( a1 G, x6 i+ ?
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
: G' z$ l0 L! `& N6 A, C19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
; b/ G1 z$ @# I* l20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
7 l) V! b0 ~ W3 a2 E21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
6 d! o. t, F, h$ J7 v& E; g22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
3 l. W& L! X! [5 [0 m( k5 c, B23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics8 y( y* l: d* u) S! p! t" `
24. Unit 4.23 Interference Reflection Microscopy
. m4 J9 C" q" k8 i25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach/ N# e/ b# s' W3 Z
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches' I# `8 }: n7 Z
27. Unit 4A Organelle Atlas: Appendix to Chapter 41 a5 K4 G% t* r1 `
7. Chapter 5 Characterization of Cellular Proteins
4 u+ @2 R W+ W7 a3 l# ? ]1. Introduction; t% j, q( i: J, d3 o, d
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells& I6 U' I z S. G7 n& e: F
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
; u+ U3 _3 [7 E% `4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients4 f' u! o5 H; B# [
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
7 m0 u+ G1 Q; d& h$ b/ j: A/ T6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)4 T; T6 w7 I% d2 `- s# Y+ N
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
. e2 _, k; I% `, ESpectrometry
9 H& j/ ^' M0 n; }2 c# P& p2 Z9 u r8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening9 Y# f% n' C/ f+ c
Applications& Z0 i }( j$ o( o
8. Chapter 6 Electrophoresis and Immunoblotting, C& I9 h5 w8 G
1. Introduction
" J$ h9 F f2 u2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins0 F1 j/ B+ _' v/ ~, Y( N; Z- c
3. Unit 6.2 Immunoblotting and Immunodetection
; c J$ g7 I |9 N1 T# z4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots# L# V1 B* I# t1 `( D; ~% P& G- G. z0 d
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
! L. m# F g* O1 o9 U1 p6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions: d) W/ m" m% O1 }
7. Unit 6.6 Staining Proteins in Gels
j7 p/ n0 ~/ U* V3 R4 K5 |8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
( E5 K2 i+ B* }( `3 L9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots( E: F$ `4 O$ U8 D, i9 O) B; C1 r
10. Unit 6.9 Digital Electrophoresis Analysis
1 }* H9 v# \3 ]1 D0 k) \11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
U! [2 \! a9 ^% W. ~5 \12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of6 r- o' I6 V3 s1 s: ~# v
Single Cell Gel Electrophoresis (Comet Assay)
$ I2 V1 w& b( b2 `0 I* K$ \9. Chapter 7 Protein Labeling and Immunoprecipitation
9 M4 D( D% \5 G% {- w+ t) f1. Introduction
/ k% a- j2 ~' Z( p2. Unit 7.1 Metabolic Labeling with Amino Acids- Y) r2 s, L+ q: o6 K) L
3. Unit 7.2 Immunoprecipitation) _3 L1 ~# v3 ^8 o8 w- |" ?
4. Unit 7.3 Metabolic Labeling with Sulfate9 R: Z- }, s* r( \ e+ o
5. Unit 7.4 Metabolic Labeling with Fatty Acids) W4 u- Q: a/ T ?$ \
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
' c: G, j& f* p" C1 @" m) w9 u7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins# H- ^; N) ^: i5 e! B
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins1 t; j, |& @( B) r1 d$ \
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars1 Q. d9 A( a* J
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
& h" {- a& k; t( d" `11. Unit 7.10 Radioiodination of Cellular Proteins9 W: |* t* d% K2 }
10. Chapter 8 Cell Cycle Analysis
& B% a& Z& K a" B$ L& u3 S9 {8 a1. Introduction9 V7 ? h9 Z* B& ^$ R9 Z
2. Unit 8.1 Overview of the Cell Cycle/ S8 `+ A! |( ~$ i1 G0 _) y
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle9 L& X& u5 ]3 v
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
- p; \# S u3 q) L8 G5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
( K* F( w+ d0 M# H$ Y* _* z6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
6 c2 K- v2 K1 g3 e1 f: Q7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
! Z; A/ ^$ y- Q L0 g11. Chapter 9 Cell Adhesion W% h. {0 v8 d% F2 T
1. Introduction+ S* K% \0 w( T5 D( B# x6 d+ i
2. Unit 9.1 Cell-Substrate Adhesion Assays
, M7 C5 N, X& z6 d2 U/ T3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force" f* S" M( M) }. M1 A, `! l
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion" X# H3 j4 i5 a7 I5 q. Q
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
" E& X$ {4 t6 o6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules3 c, M: q/ J/ L% ]" p' X: A0 F2 N
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
6 m, Y) }" o( P7 W Z12. Chapter 10 Extracellular Matrix A8 Z$ J. }" D" [2 N3 U1 e7 n
1. Introduction0 V5 R$ |+ F, c. i) ?3 z
2. Unit 10.1 Overview of Extracellular Matrix) m' E3 p- W1 A1 v q
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors! g# k9 }. v% h: C
4. Unit 10.3 Preparation of Gelled Substrates5 y$ u L* a; o6 L- o- d% R
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR90 q$ s5 z7 | m9 ?9 P( ~
Endodermal Cells
3 A- M8 }' M: ?9 {. N0 u }6. Unit 10.5 Purification of Fibronectin
- a1 x% U, G! Q: {! a2 a( H7. Unit 10.6 Purification of Vitronectin4 K9 t% l7 y! b5 P( l5 f) a
8. Unit 10.7 Proteoglycan Isolation and Analysis
* d! r, H5 G2 `$ }9. Unit 10.8 Matrix Metalloproteinases
$ J( z, x4 _) U7 L" ?6 D10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts7 t, ?5 E/ a( m# W2 z1 K2 x3 }
11. Unit 10.10 Purification and Analysis of Thrombospondin-1! [. z+ t* P5 e6 N& |6 ~; ?' E$ h
12. Unit 10.11 Purification of SPARC/Osteonectin
5 ]3 P! {5 ^3 ^" E$ V2 R% T$ K13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
" C: I h9 F- ~5 X( Q: G% i( y: ?14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly& Z" Q" R! h' g% |9 ]
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor/ n6 }& ] S# J3 ~5 y {- Q- V
Xenografts$ G2 }2 o: L5 r6 p! k* s/ n! \( C1 H
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
2 M& ^2 o# f7 g6 G17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
0 w: N1 q' M1 L% V18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
7 B0 I& D' _' F, F8 `Cell Migration# F0 n @* T' b! W1 G+ V
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
# B4 m* N0 X' v1 ?13. Chapter 11 In Vitro Reconstitution* {- o; o' N( @0 X. c1 a
1. Introduction
" H" V) E9 `. `2 R. k/ w6 O% ~2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
5 g! C7 O+ u4 t7 ^& z" u3. Unit 11.2 In Vitro Translation
1 }6 B/ d# t* J- s) b* l4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells! h( f5 b6 c" B7 X
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes' y0 a$ J$ T& ?1 y) C0 A/ k$ p. M) @
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
% @) D6 Y- M1 D' T7. Unit 11.6 In Vitro Transcription* W4 P' N7 X9 r, N/ s
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells4 @( i! I% m: v. F' a3 F4 P6 G
9. Unit 11.8 In Vitro Translation Using HeLa Extract
# C/ Y0 a% g" T5 {, [) h( z10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
( a( O1 I/ q& S7 Y* x; T" C11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
) s: u& l4 q& }' U+ X12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
4 U0 F+ c2 a y3 T0 U6 W13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
6 J7 _& v- f; u9 J; W14. Unit 11.13 Mitotic Spindle Assembly In Vitro
6 o* L/ [# q s h2 i15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes* Z# m3 g2 r( d5 B4 h
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import$ K0 f" Z* }, o
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import9 {* C y) e" H* q
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
+ b+ ~+ |% y1 M+ G" ^; l6 g19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells0 ~- h3 ]/ g4 o& O: a8 } y9 i
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import. K% a0 r: h3 ^% m8 h! }
14. Chapter 12 Cell Motility
: V* K! } G4 f3 [1. Introduction* d9 m- v [! R. I6 v
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
2 i5 I. l H# @; w" Z3. Unit 12.2 Invasion Assays1 k: M# F2 \+ ]! `! H4 i( R
4. Unit 12.3 Cell Traction
# o; o5 Y. m/ ~/ O5. Unit 12.4 Cell Wound Assays! ~+ h) D8 y: P* ~
6. Unit 12.5 Dictyostelium Cell Dynamics: ]* C: U3 J4 k4 [. f8 W
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils$ h8 S6 e* k6 p) z! B" `
8. Unit 12.7 Actin-Based Motility Assay
9 `- ~& I' x1 ^7 t' r; e7 Q9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP. J. e5 G1 V* Y4 F
15. Chapter 13 Organelle Motility7 x% k. L% u' O; K) L! N. Z( M" O
1. Introduction& R3 ~6 H" G( e/ \ J
2. Unit 13.1 Microtubule/Organelle Motility Assays( Y% H5 A5 [( l) ?5 y0 r/ y9 a) p
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
5 N! |; _2 x3 ]4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP8 v& w, J2 B. z
5. Unit 13.4 Movement of Nuclei3 i" s$ {( P% {; ~( k, f
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching+ [8 d6 N" j' N7 F9 _ U6 } S
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
: X+ w6 o" z8 x1 h' i+ e" W; M16. Chapter 14 Signal Transduction: Protein Phosphorylation
1 K9 U" f" V1 g& ?, J; D1. Introduction3 F6 e. i6 A4 @; ]
2. Unit 14.1 Overview of Protein Phosphorylation4 \+ `" _( I& C
3. Unit 14.2 Immunological Detection of Phosphorylation% r7 i+ n; }# s% \4 x* Z
4. Unit 14.3 The Detection of MAPK Signaling0 U9 l# e$ o% y6 p, I
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation4 d( L+ O8 b7 a' M7 h Y
6. Unit 14.5 Phosphoamino Acid Analysis
+ `2 \& w$ T3 D7. Unit 14.6 Determination of Akt/PKB Signaling$ x( u$ `) y) m% H. y
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
2 J) E* U8 L. O t* l9. Unit 14.8 Rho GTPase Activation Assays. q9 W, ]) V8 v5 l
10. Unit 14.9 In Vitro GEF and GAP Assays. @1 w! M9 z6 i2 Z- P2 G# I9 S
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance0 O8 n: S/ a* \3 \& ?
Energy Transfer (FRET)" x: v g0 b8 l/ {) A
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
8 i0 q1 d% }4 X2 x) [, y13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
5 i! M( c/ f) u' F- T17. Chapter 15 Protein Trafficking9 Q7 V4 X9 S2 J' [ w9 r
1. Introduction
$ o$ K& z) b- m2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways: e& a9 d4 x' \( x6 b6 w
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
. h* k7 A7 c+ c4 F9 v; ?* z# t; @4. Unit 15.3 Endocytosis: Biochemical Analyses; t. ]' @8 a& b( `( N
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
( L2 w: ^! I2 H5 I) f9 c% k" R6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells* z1 {- w0 X& K. {$ _
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum v3 b n. J. R% ~1 x, P# j% [
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation5 t, R9 ]& H/ U3 i" ^* F( Z
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
4 g* c9 h0 ^( G! v. O- P6 s: X+ u10. Unit 15.9 Studies of the Ubiquitin Proteasome System
8 W) ]2 p+ h1 a6 e9 e& n11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network7 h! K# Q' e) P; W
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
L9 v9 g( s* t2 l5 x( h* x" s$ a0 K0 a13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells/ p3 I$ J" j, P) w4 T
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging. b( r& _2 X" D3 i m8 h z( c. e+ e
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and' ~3 R, w; n0 J5 d; d V/ p
EGF Receptor
3 {3 F' I/ L1 q% S) R. s* S) v' z16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers' E0 B! G4 O, M1 i, Z9 G
18. Chapter 16 Antibodies as Cell Biological Tools
: E( t6 Y7 E+ C( P1 \7 e1. Introduction
" F7 w2 h+ g. a. R2. Unit 16.1 Production of Monoclonal Antibodies+ N# W x* a# d4 l) y
3. Unit 16.2 Production of Polyclonal Antisera4 q4 W: ]) z b3 F1 J' Q; B2 O0 b
4. Unit 16.3 Purification of Immunoglobulin G
4 u* S, a6 ?. t6 i! N5. Unit 16.4 Fragmentation of Immunoglobulin G7 n8 w8 e( R6 ^- j
6. Unit 16.5 Antibody Conjugates for Cell Biology( F, K6 x3 x$ k. ^& @
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
7 z- K3 ~5 B/ g* U- }1 ]19. Chapter 17 Macromolecular Interactions in Cells+ k/ I W% q+ ~! ]- i$ h$ U
1. Introduction
3 w/ B) r6 g; B; J- c7 K$ L) v5 A0 }2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
, s0 m. n1 t1 B( }9 QMicroscopy
5 T# u$ n8 c' b9 C5 S+ j3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis1 }% U; D, V4 E% y' F
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins$ W5 l' ]/ P$ V, Q
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries" D/ n6 `6 U0 ?7 X! ^% r- x
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry5 P# A# {- M4 _( _5 E7 j9 E. }1 z
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors: d* i7 W( H" w, v% q
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific* V+ k" n B% o/ N5 _2 w
Genomic Sequences In Vivo
; B7 ?) E' R. b* ], x0 M3 d2 n9. Unit 17.8 Isothermal Titration Calorimetry
: I$ g0 E8 {) B- h1 Y6 o# L6 E10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
8 E! {7 s3 v% G/ e) Z; u11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
6 `1 N2 g0 V8 u- R12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
5 c" S$ K2 t2 V+ z* S& vInteractions (RFAP) in Live Bacterial Cells
7 l# ~2 e9 X5 g20. Chapter 18 Cellular Aging and Death p7 \0 N- P; n2 C
1. Introduction
9 }7 @1 A' ], s* I6 H( J: ]2. Unit 18.1 Current Concepts in Cell Death1 G) e8 b9 ]2 x; W' J, d
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
) u# t' E1 ~" p) {! f% j7 t4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria# a! @) g: V1 r2 x
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
& ]1 ^" @$ [& \$ n) U+ ~/ I( G$ x6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death" }/ R4 i9 G1 U: J4 ]
7. Unit 18.6 Analysis of Telomeres and Telomerase
, {/ | O+ Y. e3 [7 T7 ^/ G) P8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of: m. Q; @) C# [5 X# |
Poly(ADP-Ribose) Polymerase
2 ~6 |9 X6 A8 {' x2 ]$ a9. Unit 18.8 Flow Cytometry of Apoptosis
- P& {3 d0 n- Q3 W L% k) Z# q10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
6 V8 a1 t: @3 iAssay& {8 g& J& K) n( _; I1 k- \
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis, r: O% l' L8 B' c R
21. Chapter 19 Whole Organism and Tissue Analysis
" o: X1 R" v5 S2 ^# j1. Introduction2 }8 n; y, q M( W$ |. V
2. Unit 19.1 Overview of Metastasis Assays5 g3 @ @3 C# r
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis- |7 _! e# U; R# Y7 u5 A! d- i
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
/ t: U$ l' k' T" |' r/ uExpression After High-Fidelity T7-Based RNA Amplification0 v8 ?0 N7 j1 ?8 `' _, T1 r, {
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
7 W+ a; S; E/ `9 z1 C( a- _. ?6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
2 s w; g: G# C. {- M) W7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo2 A( T, [% L9 R4 l3 A; r& `
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo( s z! f) x i. _) ?
9. Unit 19.8 Embryonic Organ Culture, j" i% e1 O6 @6 c1 n( q7 k
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin( V c6 B6 I& y; g. W
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
m" A5 y: Z$ j3 d12. Unit 19.11 Generation of Transgenic Mice
9 T9 @4 ]1 w7 K- C) f7 l: d13. Unit 19.12 Overview: Generation of Gene Knockout Mice& T7 l0 V. C8 l( ?+ Y
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production# s2 k1 W z+ Z* H
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection9 B2 K* \0 z) d6 D5 s: V" Z
22. Chapter 20 Expression and Introduction of Macromolecules into Cells8 k3 I* h3 Q3 i7 G! v' r& I* L$ {
1. Introduction
# F2 \' e. I, F) p2. Unit 20.1 Direct Introduction of Molecules into Cells
' e' ]/ Y6 t- k$ C$ ~ p" Q) ?$ h3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
+ ^- D0 x1 [2 |4 z6 m/ s# m1 ~6 z4. Unit 20.3 Calcium Phosphate Transfection; H1 X( D8 a: M, f1 {- f+ j
5. Unit 20.4 Transfection Using DEAE-Dextran# L# w0 }: I) F) G
6. Unit 20.5 Transfection by Electroporation9 `2 q+ d/ o" G) [
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents2 e+ }- S) H& N3 i1 g' p
8. Unit 20.7 Optimization of Transfection7 f, v! Z0 {+ E
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System1 F( h& X( k8 T% R
23. Chapter 21 Fluorescent Protein Technology' Q* P$ P8 b9 [( e0 k6 D+ J" S
1. Introduction% u+ z( u4 o: L
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells" y! s& L" S( X# s/ B1 H
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
; m9 \0 F5 M; O8 ?! D/ k4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
9 Z- f; y P0 a+ @4 J) e% aComplementation (BiFC) Analysis
6 d3 M; P8 P ?4 l% P( B( X5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
6 Z3 n5 q: b$ { J# I$ x9 U6 s6. Unit 21.5 The Fluorescent Protein Color Palette( u: d( A0 e: ?* [
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
6 X; e+ P" I' A8 W% i0 F24. Chapter 22 Cell Biology of Chromosomes and Nuclei/ g# y5 O0 E* {
1. Introduction
2 S5 ~0 }: O6 A2 r( T ]6 l @0 [2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
8 M8 Z* {% z6 x+ ~) h. S# o9 c3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
9 L' t$ f: \; b" Y4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis! z5 X9 n6 b! {' ~
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
5 f" H D4 }) r( r6. Unit 22.5 Multi-Color FISH Techniques$ @9 f4 r/ R: S
7. Unit 22.6 Comparative Genomic Hybridization% x4 w0 R7 o% I! c
8. Unit 22.7 Sister Chromatid Exchange0 [" \! |; r+ r: r7 o
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery+ {* h m: C( s3 f4 C- P% M
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
9 I( \' ]. S; P( r6 ^7 S$ k11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
% y% ^" m, `2 ~" _2 V9 L8 f: p12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly! R; Q/ p& a. S
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells6 s" y6 d* f2 Q' k1 |0 |. l
14. Unit 22.13 Monitoring mRNA Export
" s* g& u6 a7 _+ D/ q3 `; C1 D15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
1 i8 ]; V2 E( {* \/ HField Gel Electrophoresis
. h8 ]2 N5 i3 h0 }1 y, [25. Chapter 23 Stem Cells
, v$ Z5 _; g4 @' k/ F6 W2 U1. Introduction
) ]$ {5 A, f: `, {2. Unit 23.1 Stem Cells: An Overview
8 w6 U. o& ]5 C* I1 f* G3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
% r3 w( w3 s! J) X5 [/ l3 {and Differentiation as Embryoid Bodies
7 J! o2 e1 T1 |2 D \% l& ~" N4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
4 ~* N4 P9 N) V0 kVessels
% M- p& o7 x J. \8 k5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into; ]5 O& @) J" \' D* l) C
Adipocytes, b+ K/ D; ^7 P# `8 j3 K
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation: S! |( o- _ y% T9 c/ W- w
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
/ D8 O8 j4 f+ L* r1 l5 a. b6 U1 X8. Unit 23.7 Neural Differentiation of Human ES Cells/ _9 E {0 D" x
26. Chapter 24 Lipids/ U7 H0 S+ A- M: u
1. Introduction
* l! R4 ~, p O& k$ F9 |$ E' k2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
4 X7 B' x9 b& u, {7 f3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
. ]1 s1 u; |* V- a+ g4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film1 t- _7 c+ X9 H. B0 M
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
: E" y5 M: {7 c4 o$ o$ I27. Chapter 25 Nanotechnology
& r. J: q# g: E1. Introduction
4 R8 y6 I/ n4 T3 }9 `2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
% F+ J7 S1 M; C9 }0 q! Q3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering$ R6 C/ q, k, y' L+ o/ p# Z0 {" d
28. Chapter 26 Viruses% p @; q% I5 W
1. Introduction
4 E: T' V: E6 ^1 ~% x( f2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors. P( d \# b; v) B7 b# b: [
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
7 t. O+ a8 |1 x( k7 k, x, R- V# NCell Entry$ g$ E P+ H2 p+ f4 F4 r
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection2 c" U- z; Y2 X, E& \
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
( P% Z/ S( P7 X p6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission9 Y* _( A* G; O, d
29. Chapter 26 Lipids
9 k5 X3 ^( T, d/ M5 i o7 g1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
* r) ^/ h4 M; J. oReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication- a- L: K0 Q8 x$ Q
30. Chapter 27 RNA-Based Methods in Cell Biology
+ c' U9 `: M. i5 r8 k/ i5 n6 C" c7 D/ j1. Introduction+ V8 w0 m+ r' f6 X9 H
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs4 d3 s. Q" v9 |9 f& C
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
1 f5 T+ x7 L; q3 n8 v) Y# TRescue in Mammalian Cells
, Z, f5 i# w/ T! `31. Appendix 1 Useful Information and Data9 G _4 @( I% R& b. I* [( K) h
1. 1A Useful Measurements and Data2 Y7 v% d6 e6 e$ ~) U! d c
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
" ~3 a1 c( X$ m$ ? r; q3. 1C Identification of Motifs in Protein Sequences
: k. [5 R. x7 }4 A4. 1D Safe Use of Radioisotopes1 N* k7 a4 m0 W% v W" B
5. 1E Absorption and Emission Maxima for Common Fluorophores
5 ]& Y a+ S- E7 d( z( H6. 1F Importing Biological Materials
! O6 T8 m+ A* S& z7. 1G Centrifuges and Rotors- a6 @9 M5 T$ [, A
8. 1H Internet Basics for Biologists
* M& d r0 G& S% A! ~32. Appendix 2 Laboratory Stock Solutions and Equipment+ Z6 d, i! g' N
1. 2A Common Stock Solutions, Buffers, and Media
' ?, ^5 G) A7 h; n! q2. 2B Medium Formulations
4 e) N/ W, h& l/ N2 n* z3. 2C Standard Laboratory Equipment$ w8 c+ ^2 Y8 C' C7 `% T- ~# g' z
33. Appendix 3 Commonly Used Techniques
7 f2 T& i" ]4 c6 f1. 3A Molecular Biology Techniques
0 p- h7 X; }/ R* }' o2. 3B Spectrophotometric Determination of Protein Concentration& K) Q- I! R% B# \7 T6 Y' f
3. 3C Dialysis and Concentration of Protein Solutions
1 C2 k- M' m! j4 n4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
t3 Q6 V, W2 P2 ~5 ^' Z& y0 t5. 3E Silanizing Glassware/ R6 b4 g1 r/ Q# z- x. C8 `
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
. A1 e- k- F* O k, E7. 3G Micro RT-PCR
. z8 T) U _) X" ]9 g6 H8. 3H The Colorimetric Detection and Quantitation of Total Protein. S" k6 ?; I! q' ^! C
34. Appendix Suppliers
3 T' x0 ~: k# s- Y- E7 z& J1. Selected Suppliers of Reagents and Equipment; ]0 T& _ V* q0 h- S& W' {
/ D+ E: l( k9 O; n. r. e* H
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发表于 2011-3-8 15:53
