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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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Current Protocols in Cell Biology 2010年完整版 5483页; U. _: Z9 H# B/ o
. \, Z U; z T% [' o l% QOnline ISBN: 9780471143031& |/ w. u7 t. {5 Y* A. Q
DOI: 10.1002/0471143030, v' u' ]# K. p" h8 `) H. v
. s$ I$ Y6 A- L5 qTable of Contents M7 R0 H+ Y7 R
1. Preface0 {+ I7 M4 W8 \) F
2. Foreword
d% H( c/ V3 W3. Chapter 1 Cell Culture. S) n' @/ S5 A O, |; O! x
1. Introduction
% w- W1 S9 H8 S# t: G. U" Z Y2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
X- T8 Y a& t+ O* s+ w( E/ s3. Unit 1.2 Media for Culture of Mammalian Cells
+ n% v" O$ i/ H4. Unit 1.3 Aseptic Technique for Cell Culture$ D% a) G* H- B1 ^5 N+ \' S
5. Unit 1.4 Sterilization and Filtration i8 Q0 p6 \; ^* C
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
# C6 C( z5 F* W& z/ ]7. Unit 1.6 Media and Culture of Yeast3 [; Q$ X4 m! d7 C) r# s3 S8 Z
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging% F5 Y- E, T: z- z
4. Chapter 2 Preparation and Isolation of Cells+ B+ I0 u! Z6 C! q7 P6 Q* [9 k' q
1. Introduction6 H# j& y6 G! |; e3 A
2. Unit 2.1 Establishment of Fibroblast Cultures
1 f( A1 T3 M9 s K: Y. M3. Unit 2.2 Preparation and Culture of Human Lymphocytes
4 {6 { w. }) J, D1 ~* D( {5 V6 F4. Unit 2.3 Preparation of Endothelial Cells* u* v2 E5 Q% C6 p% ?. ^
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation1 h# V& A, W) L1 @; V
6. Unit 2.5 Laser Capture Microdissection4 Y! l/ V5 r( I
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
: u& v3 m$ p: N$ k: c; k8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
8 ^3 ], P- j" h. C% E5. Chapter 3 Subcellular Fractionation and Isolation of Organelles, Y' z& m+ C- C' N7 P! Y1 p
1. Introduction, I$ K5 U. B$ Y3 s6 p' S
2. Introduction
! S3 M5 i) e: L6 r v ]3. Unit 3.1 Overview of Cell Fractionation( p# E& F1 B# L2 n p e5 }$ G. \/ t5 T! E
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
" @. q! }! L1 g3 L5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
/ Z. p. @2 S8 Y P% l6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
L. ?# E( Q, y) T! Y5 h7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient" c0 n1 }% Z: f1 ^; M# i% z* ~! |1 r
Centrifugation
% X+ q3 _0 K2 i; \8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient' y3 g8 f0 B7 F8 u. b& o: J
Centrifugation
# I1 c1 g* n# p5 s# F9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae$ N! g6 \7 c& f; A8 w& r. M
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae9 t9 h) r) t6 Z/ b' V1 [( X8 p
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient8 V7 B- W5 L! y! N$ X- D
Centrifugation, C: j9 y" h' W3 G4 z
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
1 g- F5 [( S: Z9 S13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
' d# X" v, w& \3 c' r+ P. ]* v14. Unit 3.12 Isolation of Synaptic Vesicles8 H8 Y$ T0 r/ K3 N6 G" G: h! ]
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
, c; b/ }8 v# H4 d) d16. Unit 3.14 Isolation of Melanosomes
, l( E* O, d' L% V17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation5 {1 Z/ [4 |% N- H3 \; g
18. Unit 3.16 Isolation of Mast Cell Granules9 v) v: |, Q3 S! l0 @, R3 F
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster5 g! i0 a! u( a! I% V5 B' ]( h
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas+ O7 ^+ b8 r9 T4 c6 c
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
) K- s/ `+ O4 s6 s2 J# X22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
/ d* @# S4 v4 { _3 K23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes" O2 W- e. s8 ]8 p9 W/ k
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
$ }% s6 m, e/ Q f2 {8 s+ FFluids
) U! o* W. j& ?, w25. Unit 3.23 Isolation of Intermediate Filaments6 S1 H& @1 c- D* c" I# Y" {- L. S
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
% [ _& H* x' w+ b; d27. Unit 3.25 Isolation of Myelin
' {* K8 o* Z! D' a28. Unit 3.26 Isolation of Renal Brush Borders
. B [9 X) y7 I8 \5 D29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
# M+ ]' x& ~; _9 X0 w* G/ ~Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
, I& W$ [2 d6 z) P30. Unit 3.28 Isolation of Amyloplasts
' C* v" ^& ]+ Z$ ^' V* a31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins: p; U8 S/ r3 k
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
" R' ^$ {3 `$ C& M( ^- ]0 cCentrifugation
+ m" R$ ^& I$ a4 f) k$ X33. Unit 3.31 Isolation of Neuromelanin Granules3 D" @; b& Y s
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and+ m7 n! Z; [" w) m& x) P5 B5 e" b6 ]
Density Gradient Centrifugation
" G8 I- n t/ X1 y9 d, p35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments! O& o" m$ K: Q# ?0 }. |3 D
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
8 V& i) `/ l3 u0 d- L1 D9 T% \" E37. Unit 3.35 Isolation of Platelet Granules
H7 E% J3 m5 v$ a38. Unit 3.36 Isolation of Nucleoli
9 F* J6 g6 h" C5 g* M39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins. B2 ^, \& W. K+ {$ h
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates7 L) P( g( S1 J- J# U# x
41. Unit 3.39 Isolation of Chromaffin Granules6 Q( m: c! t5 n$ G; @0 X) N: L
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
, {6 S X6 u( w' L) X/ c( C- Q6. Chapter 4 Microscopy
: {0 p+ r; n3 Z5 e8 y3 r+ H2 X1. Introduction
& l) A* a( y5 x+ G6 I2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope! m: B6 n Q5 j
3. Unit 4.2 Fluorescence Microscopy
# {+ E& H7 P- {4. Unit 4.3 Immunofluorescence Staining
3 i- ~/ l3 r4 C; I5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria- Y1 y" O! G }6 N/ K% ^2 O1 F. d
6. Unit 4.5 Basic Confocal Microscopy
1 h, g* c. F I8 r7 \5 X7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
5 q* ~" V+ f5 ^/ l) U* T0 y9 Q5 i% Y8. Unit 4.7 Cryo-Immunogold Electron Microscopy
. b9 Y* V0 s, d9. Unit 4.8 Correlative Video Light/Electron Microscopy
. d+ f) R& ^/ p10. Unit 4.9 Polarization Microscopy
/ \7 R: I" w% ^/ o1 @11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
# r; J! e% b9 Q8 m: }0 d" L12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
' A; D C5 p+ v# \. D6 D7 \6 j- a13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface/ ^ Q& @7 K5 u6 v; @2 L' U
Events; P* B. A/ v4 Y1 f
14. Unit 4.13 Fluorescent Labeling of Yeast8 d* a6 q) U8 y" v& X% b
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
* o0 K7 u9 j" D2 N) s9 n. x16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
- Q, g. A4 x" @ t- ]) q, m17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules4 |- w% _+ H6 @! T# R
in Endocytosis
1 W; r! N; T: X2 y* L18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology ^ V) F% Y/ q, M5 E; V6 C
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
U4 K: y" ?8 \) I2 P; d20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images. M4 ]9 S# z( N6 r. E# z( ]: w
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions- E* X8 n* n0 n1 A. a8 g
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
* m5 @) Y5 G' h5 S; W. ?23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
2 m% _( n# ?* `24. Unit 4.23 Interference Reflection Microscopy
0 _* _& T* A& p3 w! N25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach! n1 }2 }0 e' m, ?2 Z
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches5 v1 o# M" A. X, O3 p
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
9 g' }1 ^( `0 C. A2 o9 k9 d7. Chapter 5 Characterization of Cellular Proteins
8 Y% x, r4 b3 j# F4 T+ u1. Introduction
9 V; J( N" O3 f* M2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
4 T0 P/ D x! t3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
% F& \0 t' }; ~2 z" ?5 s; Q# j4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
D5 B7 Y+ L5 g3 W- {5 T) C* ?1 O5. Unit 5.4 Analysis of the Association of Proteins with Membranes7 Y: x: V& ~& ~1 f2 g
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
6 F3 v6 n# B2 M3 ^! L- W% B* a7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
! T# ~4 R6 {3 j' u! V' lSpectrometry8 {4 b2 l2 z; N. |( U
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening# o) s- c3 |: W( n5 G' X
Applications6 G: }' H3 w! |: Q! S
8. Chapter 6 Electrophoresis and Immunoblotting
! v$ S( ~' L y/ {7 {6 L; B1. Introduction
% v5 j P+ O1 a# t/ x! d& ?: s2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins- m# A/ o( ~' x, Q0 G
3. Unit 6.2 Immunoblotting and Immunodetection
3 I1 g1 P6 I3 D9 L ^& c9 }/ Q6 N4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots/ Z; V# v) E1 G4 Y1 h+ @
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
6 _3 _$ A3 {2 X% E+ z6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
+ c8 c# R# J) S+ V1 X7. Unit 6.6 Staining Proteins in Gels
/ g- }' _ E7 G! f. g1 d8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
7 {. b; y' _- \0 _* s1 [9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots( }/ k$ c! M0 ^- R. n) l9 ~
10. Unit 6.9 Digital Electrophoresis Analysis( B6 L: g. f* q- W1 `& o
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis. w# I8 T8 N( ~! U5 p9 l
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
- X: B1 T3 @/ O4 f+ ]. |$ t9 a' p9 OSingle Cell Gel Electrophoresis (Comet Assay)
* V0 v8 O; Z& p" I! L4 X/ ]9. Chapter 7 Protein Labeling and Immunoprecipitation
* U, b4 i( _8 @8 O. Y% L) D* m! V9 u1. Introduction3 [! D/ |6 d6 B% [4 L" v, q
2. Unit 7.1 Metabolic Labeling with Amino Acids
6 N( ?& ?/ V: A) L3. Unit 7.2 Immunoprecipitation" n; V9 d( L. e* S
4. Unit 7.3 Metabolic Labeling with Sulfate) C# ~; v8 H) k# M1 w9 B' i
5. Unit 7.4 Metabolic Labeling with Fatty Acids
: k/ [! b- p' ^. }. _2 T6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups$ N# } z* x! b7 a
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins! I- ]9 l$ C }+ v
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins( N) \1 f$ W9 }6 c1 [2 Z8 b1 |
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
3 r7 f- B/ _, Q" K1 \10. Unit 7.9 Analysis of Oxidative Modification of Proteins, k7 j# S4 n0 Q. c! \9 M3 ?
11. Unit 7.10 Radioiodination of Cellular Proteins
; Q' M3 Y9 v- ?: K J- l" a10. Chapter 8 Cell Cycle Analysis
; _1 w/ H8 L" W. {/ Y4 j" V1. Introduction2 {3 z$ f+ \9 j$ {
2. Unit 8.1 Overview of the Cell Cycle4 @1 Y+ n) {! V0 F; {
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle) |. I, K# ?6 p$ {9 p. r! X
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle! f5 b' V+ F$ J& y4 P6 l( Y7 o: Y
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
' n% _, `1 [) ?2 G) Q6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
- B: s* q$ `5 w# U% v! `3 d7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry) Z2 O7 {- [/ n& u' s
11. Chapter 9 Cell Adhesion
* h) `) b, U) d b/ d1. Introduction9 Z5 i, F$ U+ W$ P& [* p
2. Unit 9.1 Cell-Substrate Adhesion Assays6 {# ^+ p3 n3 `0 K! _1 Z( p
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force y# S5 {( w+ T, c8 R
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
7 {) K$ Y N; ]5. Unit 9.4 Analyzing Integrin-Dependent Adhesion7 X3 O3 m5 N9 V' h& [
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
( G8 B$ C6 m, a7 n! ]. {7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
" A+ D/ E4 W- h6 B5 U+ {- _, s5 }) F12. Chapter 10 Extracellular Matrix: }' E# ` O& n. R2 A1 u0 V' X
1. Introduction/ j, c8 y% O5 J4 s5 B
2. Unit 10.1 Overview of Extracellular Matrix# h c: b$ w+ ~0 @. Y+ i2 d
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors2 W# j7 h* R7 m. G- x$ f+ m
4. Unit 10.3 Preparation of Gelled Substrates( j1 |0 a9 e* @
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR97 q0 J" e' s6 t% l; f3 s; t, _
Endodermal Cells% S. P+ J+ f: @; B( x/ {
6. Unit 10.5 Purification of Fibronectin3 r. s/ C! Q8 k' N
7. Unit 10.6 Purification of Vitronectin
7 b6 J1 G" Y& Q: U- l. X8. Unit 10.7 Proteoglycan Isolation and Analysis
- ^* O- Z# c2 }2 o7 y9. Unit 10.8 Matrix Metalloproteinases
3 P6 N6 I2 j8 H- F1 h, m10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts; d0 V7 M( s& X. s4 V( Z8 x% [
11. Unit 10.10 Purification and Analysis of Thrombospondin-1! k4 }2 w! R" k/ k
12. Unit 10.11 Purification of SPARC/Osteonectin) \7 m9 l0 ]0 M$ I) W$ m0 E
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
. f3 b3 c3 s9 @; i% X14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
; d1 D% X6 }0 }8 N15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
* n. j. v5 [3 P9 \1 z$ _. yXenografts
' k! F2 i; K: a/ I% L! i; @16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning( [1 x9 `/ [( ?8 G! l ^/ Z/ ]
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties1 O6 @9 H& q$ l. m
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
( n- }% |5 e2 {7 t+ i/ |0 G NCell Migration& h; ?5 s/ a9 N( ~1 O- J
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
s1 W n, G+ C" v9 X1 S3 K13. Chapter 11 In Vitro Reconstitution" p" }: z7 q9 k3 `: j7 r
1. Introduction' e4 f, J# @) M8 n% I( Q
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
3 J2 O# H, O0 B* P r3. Unit 11.2 In Vitro Translation0 S3 Q+ X& b2 q0 o6 b
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells3 Q& g) E' y/ ^& m( h+ Q. u# |
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes$ Y9 o) D, o- D+ D' R+ K
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication8 Q( G5 d0 E$ f- a5 M6 g+ D7 O. ~
7. Unit 11.6 In Vitro Transcription" }) O/ R* `1 _
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells ^2 N, l+ @% b4 Q
9. Unit 11.8 In Vitro Translation Using HeLa Extract
* h- I: E3 |2 @1 j: d* ~10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
+ w) g& s% q1 b) V+ f- N11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 D% b/ R/ q! j! W6 f) H1 |( F( L
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts8 G# M- [* S+ q& t! \
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts, c4 s8 J. Y9 }9 m, U, o
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
7 G9 k) j* k6 T, E15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes0 F% |2 d/ q6 L \! h( d: \' u( K
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import1 r/ W& c t5 Z9 z D! e
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import2 T/ t2 l: ~% L( @% Q( A" N
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
8 U! a% V- J7 \+ f+ i0 v19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
4 E9 _ W* a% B% q20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import" o: V. A5 Y$ c. i- [
14. Chapter 12 Cell Motility
" z, p& u9 _) I1. Introduction
4 w# Y( g4 s2 x) ?. c, n2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells6 Q3 z/ k d7 e: o: S
3. Unit 12.2 Invasion Assays4 E3 R. H, k) L0 w: Y8 r. k
4. Unit 12.3 Cell Traction
7 x F' r' G3 h1 R" j7 r1 `5. Unit 12.4 Cell Wound Assays
5 u0 [% F+ G( \) c' P6. Unit 12.5 Dictyostelium Cell Dynamics+ a' n7 C/ C3 U% f
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
+ q5 L7 I) A7 r5 Y8. Unit 12.7 Actin-Based Motility Assay/ a. R, x) x0 V9 h) g* \
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
9 w4 V8 {7 L% [$ C) r15. Chapter 13 Organelle Motility
9 z, B7 x$ H: c2 P6 U( ?) r1. Introduction
: \; S0 \8 f" |5 L2 x/ E* o6 Y& I2. Unit 13.1 Microtubule/Organelle Motility Assays- t& l- M' _' ?- H
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin- J }* r# z% F' ?$ L
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP2 y$ W1 j& G9 z @4 [2 t1 @3 K- f
5. Unit 13.4 Movement of Nuclei# ~6 N! t3 V3 r2 Y3 B" N% v5 D
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching% v" `* u& K/ X) K1 F
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly+ u& o- J- J( c! H Q1 Z
16. Chapter 14 Signal Transduction: Protein Phosphorylation
2 A3 C' |9 _; p1. Introduction0 K# F7 p) u0 y6 W
2. Unit 14.1 Overview of Protein Phosphorylation
2 I: ^! A5 D* M0 b' ?! Y( o% M4 R2 ^3. Unit 14.2 Immunological Detection of Phosphorylation
& E& z8 v( W0 @9 Q& w4. Unit 14.3 The Detection of MAPK Signaling3 n$ D6 w8 r0 g+ v8 R$ U: q
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation, Q) D$ E ~ T4 z
6. Unit 14.5 Phosphoamino Acid Analysis. w! ?! l" B; G7 c; d g; _
7. Unit 14.6 Determination of Akt/PKB Signaling
/ s: N0 I! S; p$ R0 Q! h( Q8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events9 Y0 W1 B! C4 y3 n8 a
9. Unit 14.8 Rho GTPase Activation Assays3 r5 T$ ^5 W4 V# |& Q. Y' ^+ R
10. Unit 14.9 In Vitro GEF and GAP Assays
( Y& U# x. [/ v; f* M11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance- Q3 L9 C( @* `! m& W( O5 ~! Y
Energy Transfer (FRET)9 e1 f7 S8 d- \/ z" K5 l6 M
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells9 @" a6 h8 u2 S/ u1 x6 m, y
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells/ h2 U4 {9 y- P) j, M7 ]' @
17. Chapter 15 Protein Trafficking# c7 ~0 F3 B! B r
1. Introduction P8 U9 S5 _0 a$ U
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways! v1 ]1 |1 O8 E G3 Z+ m! O
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking3 p2 m/ V* F" \$ Y. }" z
4. Unit 15.3 Endocytosis: Biochemical Analyses5 h0 x5 a: s4 R/ K! q
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
) D6 i4 e. q0 e8 F& R4 B( o' S. \6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
6 p0 e+ V7 H/ C5 [; U ?+ x' }7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
# ]4 X/ Y9 H" d0 m8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
7 N( ?. S6 Y! Z* S9. Unit 15.8 Analysis of Protein Transport to Lysosomes# N6 w0 J$ d8 Y- @, [ A
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
8 D- @& b- U! r/ C* F7 t5 p11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network6 i& `/ }9 U( |6 C' D/ m H
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
; z q( P: R' w) ], f, X13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
9 a& _$ w* B6 k14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
. s' V8 F/ R3 a# A% b15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and* i1 z. d! H3 o! m+ d
EGF Receptor
/ f) r8 a1 C9 d! w' D16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers. x5 {; k6 x# ^* J
18. Chapter 16 Antibodies as Cell Biological Tools* }$ ?* ?4 Y5 T0 }
1. Introduction4 N' z( _% \0 V3 A5 o, r7 }+ a
2. Unit 16.1 Production of Monoclonal Antibodies
7 A& s# L, [$ F6 y# G3. Unit 16.2 Production of Polyclonal Antisera
6 G; I$ X, L: W; V6 U( q6 M' t% B6 _4. Unit 16.3 Purification of Immunoglobulin G* I$ \ t7 b- Y' ^) G$ t! K$ P; u8 k
5. Unit 16.4 Fragmentation of Immunoglobulin G
& k7 U: ~5 v7 j( e6 H. v: @6. Unit 16.5 Antibody Conjugates for Cell Biology: w3 `# s& |7 j I+ S9 i
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides& v2 n t5 s; Q; C* e% r
19. Chapter 17 Macromolecular Interactions in Cells
8 O0 T3 u7 E- O/ \; E# c: ?" N1. Introduction
d1 G. c a7 @! m; m2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
( H* I5 p. c! n/ UMicroscopy9 P' O; Y; {1 u" c' p+ @, P) W
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
+ h; Z T9 E- S) }4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins' y/ O1 m7 M" ^& R) z B
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
$ Y8 d; K b6 {) A* p' I( C* B% S6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
h6 k4 r, s. S1 A7 k7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
+ s; e; w" ~/ L# g8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific1 p0 p/ G2 b) k: E
Genomic Sequences In Vivo# E- C9 S+ [5 I) h
9. Unit 17.8 Isothermal Titration Calorimetry2 r2 u3 `( O$ A) {( ~, C: s
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells% D& D; I5 o4 S1 R0 v$ u. z
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking3 h, b9 y3 P a7 |" [# S) T
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein) O# T1 {% d7 k) I& M
Interactions (RFAP) in Live Bacterial Cells' D$ T) C6 R- k
20. Chapter 18 Cellular Aging and Death
d3 P: d. Q! `4 Q4 P, a' b1. Introduction" Q( x* O- m3 ?4 B
2. Unit 18.1 Current Concepts in Cell Death9 ]/ n# p W' A( O5 V% ~" T2 g
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis, \5 @9 Z$ K0 I/ ~) x9 O
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria& N4 O& Z) p0 z( I/ b$ w
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)- H/ y3 C" ^, D0 }% d. @
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
( \6 n* i+ j* K# `+ W0 g8 K7. Unit 18.6 Analysis of Telomeres and Telomerase5 m! L+ A5 ~6 y- o0 c# I( [3 i
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
2 S( ~0 Q7 D3 S H- RPoly(ADP-Ribose) Polymerase9 i) f) ^1 w8 b7 X( t- F0 \
9. Unit 18.8 Flow Cytometry of Apoptosis
2 b( P* s" [) ?/ R1 ?( [, _10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
# A* q# w K1 E3 F* l# iAssay
/ w' B; |3 {* ^1 S4 d$ V11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
# j0 S8 e& E- B( k- x2 N% V5 O, E1 R21. Chapter 19 Whole Organism and Tissue Analysis( Z' R3 X6 [4 d" u/ [% K) o
1. Introduction
4 ]# `5 L0 r4 ~2. Unit 19.1 Overview of Metastasis Assays
# T5 ~6 a; v8 h3 ?5 u3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
- d/ K4 |: M J: S' u7 e4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene7 X$ c+ u; n4 X0 x9 O `/ E7 a
Expression After High-Fidelity T7-Based RNA Amplification
6 `8 b' h; t3 r" C4 N) s9 H5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA$ o1 f/ i# O b6 ?$ W
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
" v3 h# l/ z0 J7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo6 K$ ~. D: t G; e
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
/ ] d4 I7 `( I' } F* _ u0 b9. Unit 19.8 Embryonic Organ Culture
c c, q7 K1 a+ ]& E10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin: p; V+ T. c. \, T6 l
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
/ E5 |5 F! [% W. \1 W5 _2 C/ E6 m12. Unit 19.11 Generation of Transgenic Mice
, ^# f; A: i+ [2 o' [3 O ]# y4 `. A; U' x13. Unit 19.12 Overview: Generation of Gene Knockout Mice* _& L9 W( J9 ]5 V
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production' v* K0 q% u; X5 a# o" o
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
3 i( d6 r: a+ P5 a8 u1 K" }! Y22. Chapter 20 Expression and Introduction of Macromolecules into Cells2 `9 o) y. ]2 b$ p/ }
1. Introduction
9 X: G* A3 {( B& l2. Unit 20.1 Direct Introduction of Molecules into Cells6 O& _% n' w! {; p
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System. Y2 m. d* M) F3 t
4. Unit 20.3 Calcium Phosphate Transfection! w/ W2 y0 k. Y" c! d% k1 @
5. Unit 20.4 Transfection Using DEAE-Dextran1 I% p s5 r* m' \
6. Unit 20.5 Transfection by Electroporation
^; J1 k8 ?9 x( k% Y$ g. w7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents- `+ |5 U9 E6 |# t. |
8. Unit 20.7 Optimization of Transfection% x; _, W) J1 r8 L! L8 k
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
: i3 k1 z- P1 [0 P! d9 m y9 U/ q23. Chapter 21 Fluorescent Protein Technology" G3 V5 ^% j/ G( N4 a
1. Introduction; A$ _- o! ~- C* N' z5 I
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
% m2 H6 h; R% K6 a/ w$ f3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)' u! k2 |6 K4 M6 r& S1 ]+ k
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
1 K N$ E: ]/ \2 G* U R8 xComplementation (BiFC) Analysis
% B$ L4 E: U$ u0 [! Q5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology# B" E- G, k' B# A+ [2 L
6. Unit 21.5 The Fluorescent Protein Color Palette5 B% q. c' \6 E: m! D: p
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins- b# _/ M2 s4 y+ s/ ~
24. Chapter 22 Cell Biology of Chromosomes and Nuclei
* m2 A. }, c1 S) l* O/ K1. Introduction
" E# o$ v- R5 z# P9 E! Q+ B2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis) L- E6 R5 b9 H/ P. U7 `
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
h! f5 N0 F3 X7 z; l' H, r: p D4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis5 a# d! m: b! ~2 W# P4 J
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
# s3 ^6 ?6 d6 b y5 _6. Unit 22.5 Multi-Color FISH Techniques
9 S7 Z9 W/ e1 j2 Q( ~7. Unit 22.6 Comparative Genomic Hybridization6 M0 I6 R3 P+ Z6 l* L* t
8. Unit 22.7 Sister Chromatid Exchange
F4 C V; H8 Y: i' X/ t9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery: A5 o# t: p2 H6 p2 \ L
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes' f# |5 C1 O- q7 ^, s8 I6 s
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs5 d" Z) G. r+ m- _6 S
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
9 `0 p0 F8 {! g* f, U& o6 W13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
% P( |6 \/ d5 j0 |8 ^) z14. Unit 22.13 Monitoring mRNA Export
3 D% N" U7 d& ^; d" b' F6 |( z/ d15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
V- W5 Z2 h5 Z/ {4 W7 ~5 rField Gel Electrophoresis b) o" M/ e/ D
25. Chapter 23 Stem Cells- C# |! u2 B( N! G5 Y% R
1. Introduction- |8 c. b. h& L& B8 h7 r2 {+ a$ r
2. Unit 23.1 Stem Cells: An Overview
' i, m' g' b( x4 P7 H& c, f7 W" W3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture. `" V$ o; y0 O; {
and Differentiation as Embryoid Bodies
" B0 d7 j: S5 E7 G3 i$ L: K4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
0 v- k5 T' U# ~2 b$ d& JVessels
0 s& {) \7 |9 _. b. T" y5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into! Y/ f; s. p( }' ?: Y/ u# Y
Adipocytes
6 J$ Q9 L& p8 ]1 O' O9 C" a6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation5 d# b4 V3 F5 l3 L& d
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells8 W3 c( g& p& e% e+ A
8. Unit 23.7 Neural Differentiation of Human ES Cells
* j0 Y; ^: ]# ?9 g26. Chapter 24 Lipids# n1 \, [. {: u3 [
1. Introduction
6 l D' Z. H" S) z" h0 M2 X2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking6 m8 K' O% |$ a5 r( ?& U5 |
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins% S5 o( D5 S$ @" x. `, Z: [
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
: {# w# F" w5 ` E; u9 _5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
) v( J% I: a: p6 q3 R- |27. Chapter 25 Nanotechnology
0 A0 n/ N; {! E$ N) B }1. Introduction* d4 E2 \; t, \. ]
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
# Q. M c% l0 M5 |/ L" r/ }3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering d; i% N; [; e/ j! N5 F
28. Chapter 26 Viruses' d6 I, k0 X% M. Y: o* W9 W, ?' K
1. Introduction
4 a5 c* O3 z- w2 g; [$ D+ }2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
7 l* O9 q9 D2 P* t6 _1 p0 Z( T& |3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
2 `! X! Y. E8 F8 {+ }8 A2 J, SCell Entry; T+ F6 C6 }" d) z8 t
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection. G( S# [; v: G, i# d
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses/ C, i% j& b, V4 r7 o7 p, J
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
- G* V7 q0 k: V2 U7 I0 T29. Chapter 26 Lipids' M1 j4 X+ t# e! r* {; K
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
3 |0 f) [3 I5 LReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
( q5 r" ?- n( L; c30. Chapter 27 RNA-Based Methods in Cell Biology
2 [; _3 J6 z. O* y3 a! [! B1. Introduction0 Y* B ?. v; z5 x4 ?1 W
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs" B: g7 h; S8 e3 G! O
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible7 @1 d. t& }) p2 {. H4 i
Rescue in Mammalian Cells( B5 z. q' }# W0 `
31. Appendix 1 Useful Information and Data
% @9 [2 g# U- Z' Z/ A8 ?1. 1A Useful Measurements and Data/ h8 s! r' b* v, d! t
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
6 T: ]+ J# L: J3 @9 w" G1 A3. 1C Identification of Motifs in Protein Sequences9 L& j" f! x% g. m- o1 k: u
4. 1D Safe Use of Radioisotopes9 V5 [7 H4 v: K J T; m
5. 1E Absorption and Emission Maxima for Common Fluorophores
9 f% }* a$ O3 e* F' D6. 1F Importing Biological Materials
; h' @% c+ a; q# _- } \! o7. 1G Centrifuges and Rotors0 ^& x: J* e, s3 @7 H- `
8. 1H Internet Basics for Biologists/ Z4 b$ _: A3 ~" j8 R p& C
32. Appendix 2 Laboratory Stock Solutions and Equipment
8 w+ F' m8 s3 `# I5 R1. 2A Common Stock Solutions, Buffers, and Media
1 K' W4 |4 C4 N' n* X2. 2B Medium Formulations
9 a' i& ] @/ D! C4 R" |* S7 p( @2 ~3. 2C Standard Laboratory Equipment& E0 q. `0 Z8 \3 j& x; w
33. Appendix 3 Commonly Used Techniques
9 N( W& r$ b$ l, u1. 3A Molecular Biology Techniques, T6 `6 F) S/ i
2. 3B Spectrophotometric Determination of Protein Concentration# X* y5 d+ X9 H: |& l8 ~5 @
3. 3C Dialysis and Concentration of Protein Solutions
& I" Z7 j" C) l4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
( L& j8 V' K- ]5 W1 T6 S. I5. 3E Silanizing Glassware
" l# q' o! p, F6 l9 z; ]% E# Z/ I) {6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization4 V. a* ~( Y0 b0 u9 {3 a
7. 3G Micro RT-PCR+ i/ u# N: [2 m' R9 V$ z, Q
8. 3H The Colorimetric Detection and Quantitation of Total Protein9 `* x4 ?4 E5 `: m7 `1 o
34. Appendix Suppliers
/ P, z* }4 v- s2 G1 x! d1. Selected Suppliers of Reagents and Equipment+ G6 @& t* P7 l
% R+ Z+ E& _: W! f( T1 C
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发表于 2011-3-8 15:53
