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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑
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8 _+ ^9 ?" e1 R3 f+ mCurrent Protocols in Cell Biology 2010年完整版 5483页( M& d( d$ `( I2 U0 D+ r& m
! A6 Z, {1 s1 I; k3 F- d v) FOnline ISBN: 9780471143031
2 [: U4 x' h3 \; j: a" FDOI: 10.1002/0471143030
! e, F5 v% X( O( L( e U! y: Q* ^4 u/ n$ B$ E0 d: q9 B$ W
Table of Contents* a' m: O! m( s1 ]+ d3 n
1. Preface
& Z. \; ^4 `0 g3 b5 B* Y2. Foreword4 o! A- L: a. r
3. Chapter 1 Cell Culture2 T% U3 B: \; ]& t4 J' s8 R( I
1. Introduction; w' K! u! d5 N, E: ]! O
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
) t2 e; y7 L# @' G3 J% u3. Unit 1.2 Media for Culture of Mammalian Cells3 R( k" B! M$ ] r2 M. N
4. Unit 1.3 Aseptic Technique for Cell Culture
$ g1 N# p6 D, U {. ?) `0 I. l5. Unit 1.4 Sterilization and Filtration
- {- ] i: B; C% G9 }& g! S- B6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
1 u8 W( j0 l ?; V7 K& q- m5 a1 b1 ?7. Unit 1.6 Media and Culture of Yeast; l$ g- {/ P: e9 x% n. f+ L
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging3 Z# C7 o6 k. m f: C) N3 ^3 b, Z
4. Chapter 2 Preparation and Isolation of Cells
8 A2 |0 x! w4 V$ @1. Introduction
+ B8 z1 Y4 k. v+ D2 ?2. Unit 2.1 Establishment of Fibroblast Cultures# I- G Z6 I k; j! u
3. Unit 2.2 Preparation and Culture of Human Lymphocytes- o& j) o8 y, b- X& {+ @
4. Unit 2.3 Preparation of Endothelial Cells
) v6 Y% Q% f2 ?7 G& o6 L: i! b$ x5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
K: U. W9 r! g6 I' ~6. Unit 2.5 Laser Capture Microdissection/ ]+ b/ u. j7 Y6 q+ {( F: g
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
4 ?! o I6 f3 m3 A7 S8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
2 J/ z) ]' A/ l" Y3 I$ c5. Chapter 3 Subcellular Fractionation and Isolation of Organelles8 D# m0 `, M9 @' B$ t
1. Introduction4 A- X. e4 X1 Z6 ^9 U; ?8 @% |* R! g$ F
2. Introduction
3 B D, O% @, Z; o3. Unit 3.1 Overview of Cell Fractionation
8 @$ _$ i/ `: `, \6 d% d( @4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains7 {0 V6 d0 S' O# i9 m3 i/ @9 B/ X
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
2 V8 E1 ~& z+ }9 e/ Y6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation8 K$ E, {8 z" I2 T4 o7 r$ v
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
/ H. ]- h0 Z0 E$ ZCentrifugation
9 |: F8 J7 t1 w: O5 ^, Z8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
; L$ \/ T' u9 C/ U8 P% L- OCentrifugation
! Y, R8 s9 Z; X! F4 d* E9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
; a0 n$ A$ ?( E8 ^3 D10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
7 h+ N! d9 O6 ^4 A2 P11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient; M x. h0 l) j; ]' b9 O* U' W0 S
Centrifugation, s! J% n- X8 {+ W" h8 K
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues9 f' \) R' o5 m# t9 _
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
5 y; K# Y. L9 @5 o X: L14. Unit 3.12 Isolation of Synaptic Vesicles( U6 Y) X( r$ o- ~4 v( u
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation& }, N" b* H# g8 H) ?& j/ D
16. Unit 3.14 Isolation of Melanosomes ~4 r( E7 I. l! o; Z9 S) J& g9 U
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
. d( J9 U8 w0 W) r$ A0 a7 P0 T0 @18. Unit 3.16 Isolation of Mast Cell Granules
7 e/ B& c& J! w* J% }" e0 Y19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
" k( u; R8 A1 s) e20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
: L3 A. A! |" p7 W/ k- ^3 Z21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons$ Z6 Y2 D4 e. {) F
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
7 E/ C+ z! W0 M% ~23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
+ u4 A1 m+ C2 \# ?3 ]24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
2 h# F$ ]/ F+ ZFluids @0 r- Z2 _9 x
25. Unit 3.23 Isolation of Intermediate Filaments9 f! g/ R: u" r. k
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
$ T% H3 U3 K: I5 ]27. Unit 3.25 Isolation of Myelin
! t4 l8 B6 K7 [6 `( d28. Unit 3.26 Isolation of Renal Brush Borders& w& F2 q: y7 `
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane# J1 }( B! f" }, X* [, j
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts, L t8 a) S( }9 n" v5 R
30. Unit 3.28 Isolation of Amyloplasts
5 s8 I' ^6 ]* e* D/ p31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
0 U3 v& K/ n/ b32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic+ G* S% l9 g: A5 G5 l' b, d* c
Centrifugation( \7 G6 Y$ q8 t! {; }' {
33. Unit 3.31 Isolation of Neuromelanin Granules
) N$ S* T6 E: R8 C5 Z. j34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
) ~. T! H! N- M7 ?Density Gradient Centrifugation5 ^& K' Q1 A+ I. |) ~ m9 a
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
, U* n7 i4 Z# q& p+ j, q36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation. k7 A0 y" V3 n) g/ T# X5 C; v
37. Unit 3.35 Isolation of Platelet Granules) @( j; F: \, b
38. Unit 3.36 Isolation of Nucleoli
" y: Y0 D& V$ _. w" ]39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins, s4 W; E" A0 e8 E/ b, k6 z2 k
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
1 g8 z2 |; F, X" L6 O: r41. Unit 3.39 Isolation of Chromaffin Granules# z$ N4 }: P6 G$ |& [
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines( D- n/ }! d# D4 V
6. Chapter 4 Microscopy
8 p8 q$ X+ _. B( }; b6 R1. Introduction+ G* [ h1 b# O6 N
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
" A# e. G) J; L& V$ I6 ]3. Unit 4.2 Fluorescence Microscopy2 T# Y9 O6 r" u% S6 V: x& D
4. Unit 4.3 Immunofluorescence Staining
& |; f+ C) w( C5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
" P! U+ V6 U0 C/ K9 y6. Unit 4.5 Basic Confocal Microscopy+ J4 l) l4 P# b
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues6 c: r' X( l$ r# n6 k7 ^
8. Unit 4.7 Cryo-Immunogold Electron Microscopy/ l$ ~, d5 p7 W; I! x6 X1 a
9. Unit 4.8 Correlative Video Light/Electron Microscopy
# z- X5 r3 H# A8 F0 Y+ j10. Unit 4.9 Polarization Microscopy
, W- B3 X; I( B$ E7 {5 u11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
1 X) E6 A M! \0 F12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
# Z3 ~: Q, U5 A( Q: a6 o13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
$ X ~; {4 Q3 \3 K+ ^Events+ H" @" @: n9 n2 D& P3 N, h) }# c
14. Unit 4.13 Fluorescent Labeling of Yeast5 u5 ?& V8 }, f' O2 T
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
2 J; O! C! Q `, ]- l9 I16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
7 M g% d3 ]/ G B: ^17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
5 _4 X2 j- l; f$ z8 S! `in Endocytosis
% P) Y9 w4 h8 v) s$ p18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology$ ^, |$ M0 I0 E. T1 q, C! x R
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development/ p( u8 u) f2 M n! j- D3 s1 Y+ |: G
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images$ C- f" A! A. R
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions5 `7 v6 G% P) ?5 G% B
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes& H( J8 v7 k7 r5 b" V
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics2 B# j! J# W$ H% i! A; v
24. Unit 4.23 Interference Reflection Microscopy
& `7 ?9 R6 _" Z/ Y& Z% {2 b25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach# ~7 Q) v. ?5 B4 o3 l6 Q
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
8 n# e" ~" i8 \" W27. Unit 4A Organelle Atlas: Appendix to Chapter 4
: Z3 P$ L! [% P2 G# A% i8 A7. Chapter 5 Characterization of Cellular Proteins
. y2 W% j- E3 ^4 G! _1. Introduction
- U, W; x [, S& q6 K5 \2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
j; A. E' |8 m3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
* }3 e2 v( W" ^! g3 m8 l4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients( U2 \6 q6 k) U% h4 k8 |
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
' X7 g! X% F! [4 b6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
& J1 b+ w" N l. H) W7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
$ R0 V3 E8 u( I R2 ^! CSpectrometry' @4 k$ i% h) ]. I* y
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
( }2 V; b! [0 z8 VApplications1 N; U; ]- R6 I) x; ] H/ e6 x
8. Chapter 6 Electrophoresis and Immunoblotting
# i1 O. a" l$ M* S1. Introduction' j$ U: z; {. L% Q( a1 z1 H
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
0 a% W/ p# B1 ]) G3 ^* m& E3. Unit 6.2 Immunoblotting and Immunodetection
- Y9 L( L& Z& N( @% {4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots! _, ]3 r$ ^% Y* h* \( K( D
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
& ^- {5 r" m& T. ^5 B8 d) `5 x6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
- w# C4 k7 j0 j. I7. Unit 6.6 Staining Proteins in Gels
! S# `' V$ N( z8 l8. Unit 6.7 Agarose Gel Electrophoresis of Proteins1 P) [. _ b' r/ a& B6 S3 U J
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots7 j3 h; U: N6 V+ g9 o! D0 ^: C
10. Unit 6.9 Digital Electrophoresis Analysis
5 O6 O4 }; ^+ ~& Z( u& i11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis& ]" F. B& b/ \$ {
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of6 T6 _$ w, L* s: W& M& c+ Z
Single Cell Gel Electrophoresis (Comet Assay)
# O8 f: b! ~# P+ ]( T$ c# X* r9. Chapter 7 Protein Labeling and Immunoprecipitation' F! G% E8 `" |- a$ ~
1. Introduction
4 ^: D- a* x" i+ y2 P$ u8 D2. Unit 7.1 Metabolic Labeling with Amino Acids) ~$ v x: ~( Z
3. Unit 7.2 Immunoprecipitation+ |/ t) Z; c- O( \7 l
4. Unit 7.3 Metabolic Labeling with Sulfate6 I" B8 L4 w2 l: m5 h2 S
5. Unit 7.4 Metabolic Labeling with Fatty Acids! j1 g+ k8 p( q2 k/ n. F' }, L
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups# {/ {% q6 K8 S6 s. ?
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
3 h+ _6 ~/ C. |, M- A, d8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
! n+ L' z( X) k- M9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars# B3 D8 f; B. w1 Q9 c+ W% {
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
9 K* D& n4 C: c' q0 n11. Unit 7.10 Radioiodination of Cellular Proteins+ K2 p7 `* g4 G8 ]5 u' b7 g1 ]
10. Chapter 8 Cell Cycle Analysis3 f6 u) M9 {+ ^" S' H# b! g$ U/ P
1. Introduction* ?# D$ _( O# S/ t' m9 `! y6 ~
2. Unit 8.1 Overview of the Cell Cycle
" ^6 {) g- ]! z3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
# Y- {) T6 L* A) P3 ~4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
, W* @+ i) v6 \7 j5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
; k3 j9 @- u1 G6 i9 w6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
. I2 l5 X7 g3 y2 u7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
- g% W1 X4 M' y% ]+ y! l) B11. Chapter 9 Cell Adhesion
7 ~* J. [$ W o* r, j0 @+ u. _1. Introduction$ D ^( J, ^- X( L
2. Unit 9.1 Cell-Substrate Adhesion Assays p8 M5 u% W# n) r* i# O$ n
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force. s6 U9 q# @2 U9 u: ?( S
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion8 u6 \- X+ f& P- U
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion, n" O" v7 k& k. o
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules* E; G0 b: j$ G5 ?# ]" _4 B% h
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
$ _& V. O* w9 V* G. K12. Chapter 10 Extracellular Matrix$ S v, g; w8 C: l3 D6 s
1. Introduction
5 P8 X' m- [* ]% L3 H/ ^- l6 h2. Unit 10.1 Overview of Extracellular Matrix/ Q1 B4 ^% ]; N5 Z L
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
. M; s* G! [/ x4 x, U4. Unit 10.3 Preparation of Gelled Substrates% j0 X6 L$ w" O! w. c0 {: j) |0 y) m
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9) H2 W% b$ Z8 E' e. p1 B
Endodermal Cells. k5 M+ v( G6 e) P0 t6 d
6. Unit 10.5 Purification of Fibronectin0 G2 i, k5 M8 h# `6 K$ W
7. Unit 10.6 Purification of Vitronectin
p% w! d9 \+ s8. Unit 10.7 Proteoglycan Isolation and Analysis7 X, e1 M2 P5 @% S& A
9. Unit 10.8 Matrix Metalloproteinases+ q: q& N8 Z' Z' [" _/ I) M! Z
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts1 n" ]7 Z, S9 T# o' h5 ^
11. Unit 10.10 Purification and Analysis of Thrombospondin-1( }& m" X5 ]$ m; v
12. Unit 10.11 Purification of SPARC/Osteonectin
; Q/ W# p' h! _; ^13. Unit 10.12 Analysis of Fibronectin Matrix Assembly, h4 G) h* Z' ]* N
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
, `9 o* c0 X5 A# k/ B/ @1 m15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor- Z$ a- w. y5 u' s1 H" P
Xenografts. y8 v. o. m& V# ?; P8 J" s0 Z
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning$ B! k. F: }: s/ n- M
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
9 m3 z5 Q4 Y: C7 O2 F: S) r& n0 c18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D, Z( W: G% }3 r9 ~0 }7 J `( |
Cell Migration
+ s- W* g0 z; M) l/ R7 L' f, [. Z+ z19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
9 n; w4 W" `( O2 ^2 |' q. h13. Chapter 11 In Vitro Reconstitution5 i5 x& K+ R# I7 I4 y4 o+ u
1. Introduction
5 Y4 ?& d# ~( r; G' D4 P( _: Z2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems5 A* F7 ~* l# d, k& @, C2 E6 Y. N
3. Unit 11.2 In Vitro Translation Q- R" {7 j0 O& R) a0 R0 c: l* w
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells: e- R0 Y3 V+ o( @
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes- e. B# ~( I& L7 d4 G! d
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication2 J' z: g8 W- a; g
7. Unit 11.6 In Vitro Transcription0 l X! t9 e- j$ n6 `
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
% f% j/ ^7 Z: Z1 [9. Unit 11.8 In Vitro Translation Using HeLa Extract. l; k/ b" _- T5 Q1 I e
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems( T# W6 P9 V8 t% b( V3 p8 t( X
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts J! K5 X1 ~& ^: k4 T: _
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts$ K% N+ \9 p( C) `8 x: f( l& d6 @
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts: P- n- [* o* o5 \7 C* o
14. Unit 11.13 Mitotic Spindle Assembly In Vitro" l6 Y6 [/ ]' u" q$ f( t
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes2 J1 h2 f$ c" W7 u
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import- X& J! Z0 m6 ]; a: |/ |. \ l! Q9 O. D
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
9 R$ I" z' Y! C+ \5 |/ E! A18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts" q) V) R$ Y+ w, y n
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells) z( D( E$ R" x$ G
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
9 H. n2 X% m1 T! _( x14. Chapter 12 Cell Motility. g8 b$ j! K! Y" Z" ~
1. Introduction8 Y: e2 g8 d% z& L2 v! S+ r& g
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
" |- j! D8 [* ^; G9 [: l$ ^+ V3. Unit 12.2 Invasion Assays4 h* V+ M2 a" i* _& C/ L
4. Unit 12.3 Cell Traction
# u5 h: f, X& e. P- F5. Unit 12.4 Cell Wound Assays
- Y3 B4 n8 i, X3 K6. Unit 12.5 Dictyostelium Cell Dynamics0 r, B# f2 H3 v5 [
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils3 F# {5 E/ C8 h5 \, \& @9 H
8. Unit 12.7 Actin-Based Motility Assay
, Q; o3 [! L% c+ ]+ ^$ F2 J# d$ t9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP& k! c* X3 a1 k4 M$ U3 x
15. Chapter 13 Organelle Motility
! V4 A; H5 R3 _4 W* F, x' e1. Introduction
% N7 }5 ]' d6 e4 ?2. Unit 13.1 Microtubule/Organelle Motility Assays) x9 X( F& c; _' L4 [
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin2 |% Q( ^0 A( Q% s7 X; C
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP6 u6 I/ m! M! V- Q- A$ e
5. Unit 13.4 Movement of Nuclei/ P! S, O T: Y9 @* P, |" S
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching6 }& ~6 ~7 ?" r' x0 n8 f
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly. ^( Z) ?: c& F# b' C, n
16. Chapter 14 Signal Transduction: Protein Phosphorylation5 {* T `' {; q) S& k/ k
1. Introduction
^* w' I4 _& [' N2 G! P5 F! k4 t2. Unit 14.1 Overview of Protein Phosphorylation
8 F, U7 y' C0 @$ ]3. Unit 14.2 Immunological Detection of Phosphorylation+ i1 M$ n( M6 @; \- q6 p9 j# [
4. Unit 14.3 The Detection of MAPK Signaling
! B* `' [% z* n5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
: X! u% ~ Z& r( E- d1 W) Y+ b6. Unit 14.5 Phosphoamino Acid Analysis
+ f) y. c1 z+ u' Z. F* {) m8 G$ J7. Unit 14.6 Determination of Akt/PKB Signaling+ g; V) m f8 I1 t
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events: S% P) y2 P' d9 t
9. Unit 14.8 Rho GTPase Activation Assays% H% r0 s3 m' W% t
10. Unit 14.9 In Vitro GEF and GAP Assays
; |$ b: L, ]& C/ |11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
2 y5 C* ?+ r3 J" z4 cEnergy Transfer (FRET), m+ i; q/ X: J1 V( X( r
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells! x, e" R$ _2 x! _
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells( e# z9 d- ]) Y/ o+ J, U4 B
17. Chapter 15 Protein Trafficking
( S5 H1 ~" \. a. U8 z1. Introduction
( R$ a q) T' u7 W* Y/ @2 o2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways# U2 s* z* ]" F o ~3 N
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
' ` h2 e1 \; q: Z3 w4. Unit 15.3 Endocytosis: Biochemical Analyses
2 i# k2 B4 u4 [5. Unit 15.4 Determining Protein Transport to the Plasma Membrane9 x% ]/ q4 f" o8 D7 ~( |
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells. H& |8 o( P4 G/ i' G
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
2 Q. C7 y0 Q8 n2 N' }. y8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
5 |- _( N+ }2 b$ w9. Unit 15.8 Analysis of Protein Transport to Lysosomes
9 K* y* U [0 j2 x" r7 h: U( R! T10. Unit 15.9 Studies of the Ubiquitin Proteasome System8 H( Z$ ^% { T7 E
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network' x# B3 J6 Q P& c3 y+ X6 h+ }
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
! U' G3 ~- j/ K0 [. \$ X13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
( s6 _: c3 A X. L; a5 Q* I* k1 v14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging. z+ N8 M# R4 j" e
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
5 r- E* s/ b; q$ F) N ^+ }EGF Receptor
$ ^' L# U* I% W/ T1 O0 U16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
6 B) N; D( K; \$ ?/ I18. Chapter 16 Antibodies as Cell Biological Tools3 E9 h% N2 X/ k) B7 E
1. Introduction/ K) a/ h& V- ~% T' |+ k) Y
2. Unit 16.1 Production of Monoclonal Antibodies% @7 s1 L. n- e$ g+ c0 @
3. Unit 16.2 Production of Polyclonal Antisera
6 a/ i# O7 }+ V4. Unit 16.3 Purification of Immunoglobulin G
) f* J1 ^2 g1 l6 s, K* v/ W5. Unit 16.4 Fragmentation of Immunoglobulin G
9 } S7 n, U+ A' N7 F. H0 e0 ^6. Unit 16.5 Antibody Conjugates for Cell Biology+ b* i- d' o+ Y' I
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
% g" K$ \" ?3 D& B5 `* s7 r: J4 M+ E19. Chapter 17 Macromolecular Interactions in Cells/ g/ u( O$ P/ K
1. Introduction
6 L$ ^$ b& p G" l, f, ?; G2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
7 b4 J/ B# V3 w! ? n7 SMicroscopy
2 j/ q/ B% A) s+ E3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis/ o7 w6 u; @/ N% y6 U
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
" G5 V7 z% A4 r( d1 T. |, T5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
0 p5 i7 I, ?( w' e8 Q6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
8 k. K* n5 p# i, `7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
9 Q; v5 l1 X; v& g9 U8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
( L9 E2 E6 x4 ]7 ?! GGenomic Sequences In Vivo- S: W4 l! X F
9. Unit 17.8 Isothermal Titration Calorimetry# N q j/ R$ v! Q# S' k
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
; G+ G( T. s# l$ ]8 c' {7 t! A11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
1 T9 ?3 i/ D- ]" x; d12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
! H0 w' s- q, U* i0 }7 SInteractions (RFAP) in Live Bacterial Cells( s& E* n6 n8 x) U. z _
20. Chapter 18 Cellular Aging and Death3 \, b# Y L5 c& H G, m6 o
1. Introduction
4 k5 X; {$ x# B( p& _2. Unit 18.1 Current Concepts in Cell Death( ?: |7 p8 k: L+ }3 }$ \# `
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
) K& y4 }: c7 B$ U4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
1 n6 N, d& T* T5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
$ m7 T2 J( d( @. X7 e. L: \6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death& l& [! [3 y6 H) B) ?6 v
7. Unit 18.6 Analysis of Telomeres and Telomerase7 ^8 H2 @% A; G2 l1 P7 K, ? D
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
/ J5 p* |: v3 R+ u P* zPoly(ADP-Ribose) Polymerase
2 L6 x$ z; {' Y3 a9. Unit 18.8 Flow Cytometry of Apoptosis; B% d: w* J# \
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
! V: l, L( V# w& i& u1 AAssay2 C& g" ]) |9 G% h3 K
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis9 `+ @9 K1 y; P$ N
21. Chapter 19 Whole Organism and Tissue Analysis
( g8 @, b- } b* k+ r2 }* \- C3 G' G5 K5 u1. Introduction
* A" H8 ?+ G+ d5 {8 D' S/ i2. Unit 19.1 Overview of Metastasis Assays
4 l: O+ h0 e& I& E* Y3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
, o4 O% B t! d, `$ t% d# _- @4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
- t5 h- W @5 ^8 U2 U$ uExpression After High-Fidelity T7-Based RNA Amplification+ n" U! T5 d1 i& ~0 g
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA4 w1 n" E: O2 X: n; x1 z# }
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model i. B# g- `8 b0 E5 f0 k
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
0 m$ U: |4 V+ s7 Q1 j8 J8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
' U, D/ s+ v5 L: D9. Unit 19.8 Embryonic Organ Culture
* y" A/ Z+ {( ?" _6 W5 y+ t10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin T$ m( q: K. r9 |( M* p% j' B
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice5 x: F& G5 h! Z9 ]
12. Unit 19.11 Generation of Transgenic Mice
+ g$ o+ B( X/ q# h13. Unit 19.12 Overview: Generation of Gene Knockout Mice4 U1 d* J5 r1 M% F5 z) H
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
7 A) o/ R# z$ [# e15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
. m. c) P8 g; m1 y- ~22. Chapter 20 Expression and Introduction of Macromolecules into Cells
2 ^6 C% Z, d) w1. Introduction( M" w0 I, `/ P+ h7 P# k
2. Unit 20.1 Direct Introduction of Molecules into Cells
6 }: Y! Z* I2 S. j6 x1 a( ?3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System8 R; a7 Y P' u- t2 h# j/ y; a+ d
4. Unit 20.3 Calcium Phosphate Transfection, R5 x5 ?! R7 e( N1 R
5. Unit 20.4 Transfection Using DEAE-Dextran" D. \& C, N+ F/ d4 K' j5 e
6. Unit 20.5 Transfection by Electroporation
: p( m: q6 O" d" @/ z' d1 P+ o7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents# P# Z( n8 r( Q4 w/ _6 v
8. Unit 20.7 Optimization of Transfection2 c7 K* F5 r4 ~0 q. _8 g: S$ }
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System# g4 b' l4 }5 T5 U1 t
23. Chapter 21 Fluorescent Protein Technology. S5 q- k! `, B. Y9 f6 N
1. Introduction: {9 F ?8 G/ h! D. b8 H
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells" _. z, @* ]2 i4 z3 j$ x
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
$ F8 `! y5 S8 ~# ^1 d. G4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
+ |% x8 k0 `6 h" ]Complementation (BiFC) Analysis
: _$ | v: E4 E" ^9 x6 Q5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
! ?5 u: `+ ^5 c' w$ w, ]( ?2 s8 }6. Unit 21.5 The Fluorescent Protein Color Palette
) x1 Y/ A, l6 x |. v/ W. {7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
I6 |) J( L2 g' s* G24. Chapter 22 Cell Biology of Chromosomes and Nuclei7 }# v) x% m5 A9 R: H" n
1. Introduction* V4 u) j1 s3 E
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
0 n9 o0 C# V5 v- l3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
% I+ d& |' j6 v5 j R# T. U/ `4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
. w8 [7 Z7 g/ E0 S$ H) v5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)! V& V' i8 o7 j
6. Unit 22.5 Multi-Color FISH Techniques1 G- j, ^& `; T) C- L
7. Unit 22.6 Comparative Genomic Hybridization- c4 x/ A- ]7 Q$ m, A4 W% w( x2 g
8. Unit 22.7 Sister Chromatid Exchange3 o. F- W% a- Z% J* l! E0 R+ P
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery" o3 L$ q* X$ H" x5 g$ {: e
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
, d3 O! X" Y' ~; w11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
+ I! N# Y3 W: K+ n8 k- b12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
4 X9 J, B7 _- ]7 L# \13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
3 `! ^3 C u1 U! [- `4 ?$ G; m14. Unit 22.13 Monitoring mRNA Export1 F- }% \5 ?4 j$ u# r
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
9 y+ P; n$ s4 f+ X3 @Field Gel Electrophoresis! h4 N" d! _. g- Y, U
25. Chapter 23 Stem Cells
# j( H# {/ y" Z9 Z7 H2 [; C8 _1. Introduction
8 S6 M; t) ]" P2. Unit 23.1 Stem Cells: An Overview3 L8 o$ I7 Z: n8 T& r2 ~
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
x+ k; n% _4 P0 ~" oand Differentiation as Embryoid Bodies
# Y& V, m! t h2 E0 u! ]6 L5 w# E4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
8 `+ B& T- G8 C% `% M! @8 LVessels5 p/ w; s( C% K9 T# k" Z
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into9 K5 @! s2 M- t* U
Adipocytes
5 b _+ o5 j4 M% ]6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
9 V$ H# _# M: j% [* E& {7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
- _3 W3 e# R9 i5 c4 `0 V8. Unit 23.7 Neural Differentiation of Human ES Cells
& k- e& C1 i" C5 R4 Z e& H% Y5 {26. Chapter 24 Lipids
# b/ v) h1 v5 L& k7 x' \1. Introduction
- B: ]. V) @# C0 I2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
" k: T- S* ]) u, |! F2 ?. ~3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins" p+ x' r5 }8 n( F# O
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film2 ^6 J1 g/ e3 L0 M- i/ B/ M
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains$ y' [2 r) P& D$ k
27. Chapter 25 Nanotechnology
4 O; [: \. p# E3 u1. Introduction
+ T1 f+ \8 F. Y f" p& T2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
- x) Q( ], ^( F7 R3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering+ e$ d4 A' i& h2 t- W: T
28. Chapter 26 Viruses# q$ g$ c% y2 {& O& ?2 ~. p( k
1. Introduction1 B9 z x& J" I/ i' B
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
+ |9 O1 w3 h L: R2 D- L, x' J% g3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of' D) W8 m" b* o; l* x
Cell Entry
1 d) q4 m. G4 W& V. s: b& \% B$ j4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
. ?3 N9 J' A+ G9 e: x5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses0 k( w8 h2 s! T4 p1 Y- m
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission# @; H' N: c% o% ?, j' C
29. Chapter 26 Lipids/ ], z9 Y" p2 c5 S
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
- p; U1 F! p# }8 ]: u5 }1 N, BReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication; l8 L0 s- k. P6 P+ c2 m
30. Chapter 27 RNA-Based Methods in Cell Biology
5 g' u2 t3 u& @) R1. Introduction
/ p. e; O+ r m2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs9 S5 H; s" [ i; y6 k) |9 I) v
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible" s0 s( F$ `9 O" O
Rescue in Mammalian Cells2 C% ?: F! ~/ J z. K) l! O
31. Appendix 1 Useful Information and Data
/ p$ S7 n9 T4 E0 v6 D. i! q1. 1A Useful Measurements and Data2 I+ e* G- w. ~. I2 c$ {9 r' h6 N
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
' a( \! ^( K g; c3. 1C Identification of Motifs in Protein Sequences
7 Q$ y- _, }3 S4 U% ^4. 1D Safe Use of Radioisotopes1 t/ Z" @! Q9 f/ h% ]
5. 1E Absorption and Emission Maxima for Common Fluorophores$ O( K# t: }# R
6. 1F Importing Biological Materials
4 H* n0 I/ i- ]& S; l+ A7. 1G Centrifuges and Rotors7 g% v7 ]. }8 ?; |8 x
8. 1H Internet Basics for Biologists
6 R$ B1 F, x. Z* x% d0 O32. Appendix 2 Laboratory Stock Solutions and Equipment
- ^% X" Q( K2 E& F, x- p z1. 2A Common Stock Solutions, Buffers, and Media
' T; N( o4 X! K% Y0 n2. 2B Medium Formulations
3 X3 g" D1 U& r2 j8 _1 U; j3. 2C Standard Laboratory Equipment
8 b( d! Z) j; J" j1 T33. Appendix 3 Commonly Used Techniques
& j3 E$ ^& ]8 Q) i. Z' C$ b6 _1. 3A Molecular Biology Techniques
4 h' L' p k5 s5 Y2. 3B Spectrophotometric Determination of Protein Concentration
1 O0 v @- N' y ^3. 3C Dialysis and Concentration of Protein Solutions
/ j" J3 i, ~$ j, d/ o* @" P3 @% m4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy9 B. O* ?$ P2 g. A0 {5 Q
5. 3E Silanizing Glassware
. z" f# Q6 d6 t/ S+ Q4 J6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
$ x4 H2 j9 g- {% V4 e( j$ D' j7. 3G Micro RT-PCR+ u. E5 Y+ M4 K
8. 3H The Colorimetric Detection and Quantitation of Total Protein
$ A' F% D" ^0 ^9 B ]34. Appendix Suppliers+ O [ F7 ~9 g5 N" z6 a2 M
1. Selected Suppliers of Reagents and Equipment1 J9 e" a/ c1 P" P7 ?- Q
- S: U- [5 e! o1 a6 X6 Y |
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发表于 2011-3-8 15:53
