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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 5 u8 n# B9 _' N5 O
: ^* s8 V8 U% a5 q; c* h: m% SCurrent Protocols in Cell Biology 2010年完整版 5483页
5 Z6 c' _. p1 H+ x, D3 S7 c$ P6 d; M3 h- C
Online ISBN: 9780471143031: |) o/ q, A4 P
DOI: 10.1002/0471143030; C6 I# R" o& B0 D, v
4 l. S, ~- Y* Q
Table of Contents
. M% D& x4 A3 q7 D* ?) h1. Preface
$ e/ i4 A; f5 \0 M2. Foreword
# I1 ~" o& {) e+ s3. Chapter 1 Cell Culture; D: n6 `7 I! Z4 l8 A
1. Introduction
/ N4 H; { e$ R! _ t' J+ V$ a2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture% g7 }& |9 j! y$ @+ @3 u! q8 i
3. Unit 1.2 Media for Culture of Mammalian Cells0 B" S4 [& p Q4 n6 G# J6 p
4. Unit 1.3 Aseptic Technique for Cell Culture
7 O1 S2 U; o6 T$ Z. I2 ^2 d9 O5. Unit 1.4 Sterilization and Filtration
* R1 M0 Y( ?2 P6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures5 Y* d5 A# C. ]4 t, j- x9 d7 z. E" j
7. Unit 1.6 Media and Culture of Yeast% J4 W7 G- y! M+ D
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging/ E: _ N6 ]" ]3 w9 L9 D
4. Chapter 2 Preparation and Isolation of Cells
" a* A* P/ I2 X# F: Z! s' {( e% s1. Introduction
/ K1 n5 j7 Y X* ~2. Unit 2.1 Establishment of Fibroblast Cultures/ w5 }6 {. F( P* m
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
/ N# h7 T$ y3 G% }' a4. Unit 2.3 Preparation of Endothelial Cells+ B8 c7 U. b. M1 N
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation& C- x. |* \+ l6 F% ^
6. Unit 2.5 Laser Capture Microdissection
4 N% ~0 B0 R- c* M7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
5 Y) s8 W; k1 k3 g; y% h) ~% o6 U9 l( ~8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
" J$ |. t7 Y2 j6 J& r5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
) G. W7 J* n" ?- |+ z1. Introduction2 O* M, j0 b! R" p% t
2. Introduction
) r) |4 L/ d" v) g6 p5 }3. Unit 3.1 Overview of Cell Fractionation! \5 h5 p6 @1 Q
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
; S5 ~5 `2 A j+ e9 `5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation2 D) l% D1 `4 i, O, w: i b+ a7 K0 A, P
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation$ m" d, B& P" \) ^* j8 Z
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
. p0 J4 H% A7 _! x. E% J; iCentrifugation
6 M. N5 Z. }& e: n% ?5 P( c8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient4 r ?% }- x: Y; D5 z
Centrifugation
! Y0 e$ H, @/ X3 \' @3 M9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae" r% a& M \3 q5 b7 R+ D! x- ]3 S
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae' g/ q2 q! `$ y6 p" m# k
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
1 W0 z) z) t+ t, XCentrifugation9 R0 A2 q1 m0 S7 i; y/ z
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
8 J2 `3 @; M X3 a T& e( ?, L- X13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes+ c8 z# A% }+ j. t
14. Unit 3.12 Isolation of Synaptic Vesicles% J0 z7 [' h3 P2 l
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation5 R8 X8 J8 y) [$ S- D' p2 l
16. Unit 3.14 Isolation of Melanosomes
: y; x6 h4 ?# p4 p6 p17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation7 j2 B+ c1 e' |+ P8 L/ t4 A- X4 w q
18. Unit 3.16 Isolation of Mast Cell Granules2 r$ P8 o# B0 t9 \( u
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
& B3 g0 c; R3 U& @% }20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
% k2 \& E7 \( o21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
( o6 j9 T5 e4 x0 K9 K. ]# P. F4 X22. Unit 3.20 Isolation of GLUT4 Storage Vesicles6 t1 d8 @4 ?+ F+ t) X
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes1 w" K9 \- X4 H; q/ O1 m
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological. b' `5 D- V7 A* [2 n
Fluids" J0 ?/ E8 T+ ?7 i
25. Unit 3.23 Isolation of Intermediate Filaments
( G3 Y0 r5 j! g/ R+ {7 c26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle h4 g2 F% f9 u$ T
27. Unit 3.25 Isolation of Myelin
$ W- Q1 i( ?. z' J9 c! w) F% S7 }, q; ~28. Unit 3.26 Isolation of Renal Brush Borders/ d8 q. S+ u5 c3 Z
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane/ f7 ~0 T7 x# ~3 ~; y; |% r
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts3 ~! A- U# [) r$ F! f" v; {
30. Unit 3.28 Isolation of Amyloplasts6 M. k+ M4 @# k5 q: v ~) Z
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins# r, d. m& F1 G6 i/ l1 ]; N0 ~% q, W
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic ^3 z1 {7 D8 J1 f
Centrifugation* Q+ ?) C& \$ _
33. Unit 3.31 Isolation of Neuromelanin Granules
, a( }* G q/ k+ |: z( {5 H34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
( q. R7 w: R8 g) K( CDensity Gradient Centrifugation( O, p0 H5 X% e5 {# `' b' j
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments- G! D' Z9 L2 F' l! z' e
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation. }2 _* a; y" `/ n
37. Unit 3.35 Isolation of Platelet Granules; H+ P0 G# T) o6 F
38. Unit 3.36 Isolation of Nucleoli5 [ N( @) W' k) D6 T
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
# @1 R) Q* P- J+ I i- w40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates7 G- C1 Y) t9 t: K5 o: B; [
41. Unit 3.39 Isolation of Chromaffin Granules
1 w/ J; @6 [6 ]# Z$ e# q42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
$ @+ B8 Y+ @" c! z6. Chapter 4 Microscopy
( z' C& R" L8 `% R- @1. Introduction
( o4 Y! F" l% y+ D5 p( u2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
# K6 T& V5 Q1 F. Q3. Unit 4.2 Fluorescence Microscopy
; o+ ]0 ?2 K/ a/ c* L% x$ A! Y4. Unit 4.3 Immunofluorescence Staining
. t) \& \( A+ h7 k* _; k' |5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
8 R u- b7 T6 Z! h$ O6. Unit 4.5 Basic Confocal Microscopy3 V" J7 M) ^# l: V" l
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues- y6 n# `$ @- H# d- @8 i) B1 }/ [
8. Unit 4.7 Cryo-Immunogold Electron Microscopy7 ~% c) `, D3 R; B1 F2 w
9. Unit 4.8 Correlative Video Light/Electron Microscopy" i# R0 _1 I% e& b# T- v) E
10. Unit 4.9 Polarization Microscopy/ h# g! y% T& t8 k0 z$ l
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
# B3 T" g4 o% y5 H12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues0 ]( [1 V |1 T" _0 d4 |
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface3 t0 Q ?: e0 p/ Y# v* w8 ^3 v+ T
Events9 g) j U& z7 O& q* W! ?. g
14. Unit 4.13 Fluorescent Labeling of Yeast# D! T# U" z: R# ~
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy( G, A3 P" F' y4 E+ s E9 o2 F2 u& X
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy6 @& D& k4 _* K) m# u1 r N
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
5 K) y! S: K; M$ gin Endocytosis
% u# S# j% k" O y& Z/ t: M1 u18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
9 e4 Q. D+ @# W, A7 _19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
: D3 Y" p6 w, A8 S! i20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images+ S5 L5 }1 H$ e7 i) I4 A2 u' S0 d
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions; a1 c( r0 q+ v% g2 N5 n
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes) m0 _& `' A+ `$ l' y
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
0 a0 N3 X5 U, C; o- ~' E24. Unit 4.23 Interference Reflection Microscopy$ c0 n& S" Y; ? ^, K9 C' Z N
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach$ c4 W- O8 b d6 |% K
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches# J9 n% T; C! ^* B& W
27. Unit 4A Organelle Atlas: Appendix to Chapter 4% ~) |2 ], |8 ~% R- T# ~
7. Chapter 5 Characterization of Cellular Proteins8 U; d* ?3 x7 f
1. Introduction
: A" s1 j% J) z8 n: ]4 N2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
% r8 t- P( ?% B/ `; I0 {* z3. Unit 5.2 Determining the Topology of an Integral Membrane Protein6 N4 H( U( T+ H2 P; c4 D
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
+ g. B! V1 }6 s3 z; u5. Unit 5.4 Analysis of the Association of Proteins with Membranes4 |" `/ Y1 {8 M
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)- t4 M/ h( N- l
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass' c) u2 b+ a, G- H
Spectrometry& ?6 X& o) \8 E- \
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening2 I8 L, l0 {8 K& [! y8 k5 [
Applications
W! N7 M* G( g* [0 b. z5 N8. Chapter 6 Electrophoresis and Immunoblotting0 _. [% j4 w( W
1. Introduction
9 t. s1 R% \2 K q2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins1 m- b& Q- n7 k5 u/ D7 u# g3 X$ T, i
3. Unit 6.2 Immunoblotting and Immunodetection
+ N" v, m$ B4 p4 o. {4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
: a" S% o8 e+ [+ m& U, T5. Unit 6.4 Two-Dimensional Gel Electrophoresis- `& k" h# v5 I9 v
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions$ I: Q4 O _- l6 ^
7. Unit 6.6 Staining Proteins in Gels
. J; \3 T' B3 D' F" o- S8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
" E( ]" @8 p5 G3 @: y9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots7 J' \/ L" _/ q) c6 W6 Y; \1 ~& q. ]- q
10. Unit 6.9 Digital Electrophoresis Analysis
) K: t5 { { j" E% t/ U11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis4 y+ t: c/ c' f' J
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
2 ~1 @/ k9 Z5 U6 ySingle Cell Gel Electrophoresis (Comet Assay)
8 d) G/ o @7 n0 H9. Chapter 7 Protein Labeling and Immunoprecipitation
) v9 p7 \0 |- v5 c( m9 d" }; a' M1. Introduction
0 Q# w1 k3 c+ c2 y0 Q' G% N8 S2. Unit 7.1 Metabolic Labeling with Amino Acids
, s' R1 h1 |8 O; p" t9 W+ \, L# }3. Unit 7.2 Immunoprecipitation
2 v' m- q) G ~8 p4. Unit 7.3 Metabolic Labeling with Sulfate& D/ e* P3 e# o8 b" ~6 ]9 w7 a
5. Unit 7.4 Metabolic Labeling with Fatty Acids
7 ?( I! O" g5 @+ A; c4 b6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
7 t: f/ t, W+ t7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins+ }' J# I, V$ i# f- A
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins( e8 j, r% a; A: p
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
0 v# g9 s( L8 x10. Unit 7.9 Analysis of Oxidative Modification of Proteins
6 a% N* y5 W. U. z+ h11. Unit 7.10 Radioiodination of Cellular Proteins. |% _1 K6 Z5 _, \/ f c& N
10. Chapter 8 Cell Cycle Analysis5 c w2 J* V2 y4 w6 y8 I7 i
1. Introduction
; U! t, c- N8 d) f2 h0 S2. Unit 8.1 Overview of the Cell Cycle) X: X, Z( a0 y' S. Y0 _& `
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
0 d: F9 J9 a3 ]" p4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
9 q t' t5 H- o3 B/ k2 y: U5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry4 U* R! L y& a Q; A0 S7 h
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
3 H* `% U" s& T8 a3 x! G* u N1 A- m1 t7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry& H" v( j2 z ~; b
11. Chapter 9 Cell Adhesion
$ `7 d# b5 u5 d0 o2 B! w" e1. Introduction \9 A1 ~& L, C
2. Unit 9.1 Cell-Substrate Adhesion Assays
1 A; Z+ ^: H& z/ n3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force0 L# y ^0 U. C3 k& H% o
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion6 F9 P- X# C9 u' G s% Y
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
8 z( ]( Z. T$ F5 `6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
/ k! I2 i# F/ E+ P9 S, m7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
4 ` ], F. [2 P. y$ p5 G12. Chapter 10 Extracellular Matrix" _ E0 q* H H, ]
1. Introduction' W& z8 P% j1 @% f6 T
2. Unit 10.1 Overview of Extracellular Matrix3 H2 F( J$ a: i5 j% `
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
* w: P' X/ y( j" h- _4. Unit 10.3 Preparation of Gelled Substrates
5 X: D- W" ?; A/ o% g4 O5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
9 L2 G0 {/ x9 A" f9 o; x/ n' XEndodermal Cells
" O( S( z( V3 H3 l6. Unit 10.5 Purification of Fibronectin7 Z) b& e% y" Z7 e
7. Unit 10.6 Purification of Vitronectin4 o: G+ }+ b$ K# M/ n) r
8. Unit 10.7 Proteoglycan Isolation and Analysis' O5 c2 H5 Y. ~; Z2 [
9. Unit 10.8 Matrix Metalloproteinases
2 N/ _8 }5 A( U1 j, M5 |) J10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
) \0 T( U# c7 s11. Unit 10.10 Purification and Analysis of Thrombospondin-1
9 b1 v! b( s @. y$ a$ O12. Unit 10.11 Purification of SPARC/Osteonectin' P9 J8 m, [, g/ V
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
$ H9 F8 Q& G& P14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly: V$ O* n. a i# T5 Q
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor2 _) j9 G% Z0 E* {& P: ?; _
Xenografts3 X3 y+ v6 u& M
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning+ {; w& d1 [3 f. w8 g/ z5 E
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
0 F+ H$ b3 t& d3 H; m) [18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D5 l& B2 I; [* `0 s4 b
Cell Migration0 B9 [8 U9 `2 S
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix; _/ G M% G1 \9 p1 A4 T; N" Z
13. Chapter 11 In Vitro Reconstitution# J7 u" r! R- F; c3 ^
1. Introduction5 E& I9 _) f* d8 a7 N
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems1 D1 r; f9 z8 l; s4 V3 c; C/ u" R
3. Unit 11.2 In Vitro Translation
L9 }% I3 ]& m4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells$ Q( a9 D' O6 Y& X
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
) W- z8 ]3 H! x, @6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
' c- L6 f1 l6 X9 q( J& @7. Unit 11.6 In Vitro Transcription z5 N/ r6 ?5 X: t, c2 E! m
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells5 l3 ^% s# d$ K
9. Unit 11.8 In Vitro Translation Using HeLa Extract
! k4 `7 u1 p9 x10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems1 h [8 d8 h8 {' M, H, e& I
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts' _" O6 o7 o; k5 f* W' ]% c
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
: x' G- E. T8 u7 w& \13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
4 B* l. i7 S" _7 Q, h14. Unit 11.13 Mitotic Spindle Assembly In Vitro
, w& X# O' w, |. S- T2 M15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes4 x# P/ W/ ^2 G0 y9 s
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import4 M( Z+ e3 d' O
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
; }& d/ v4 Z6 H9 @. H18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts% K" l/ i9 }. p- S
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells d3 `/ i* H! E/ u0 U
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import. j/ N7 z( z$ s. \( K2 A. G
14. Chapter 12 Cell Motility {! A; `; z; F6 ~, `: X/ H: ~
1. Introduction
8 t* b) h! t) \% t8 |2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
: I+ q& _+ K5 i3. Unit 12.2 Invasion Assays
: u/ x+ E4 S& R/ ` L+ A) K9 c4. Unit 12.3 Cell Traction
* U) H. L, v2 P3 d. U5. Unit 12.4 Cell Wound Assays
/ a7 ]6 i. w- q5 u) m9 k* Z6. Unit 12.5 Dictyostelium Cell Dynamics& Q+ w5 f& U4 ?+ D) W. ?+ j- h
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils7 p, q1 m" l- Z
8. Unit 12.7 Actin-Based Motility Assay+ X( j. S6 T3 Z
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP) p' m! Q3 G ^
15. Chapter 13 Organelle Motility3 w( h W' u- [; O) g' v
1. Introduction
3 z5 h# Y% C5 d2. Unit 13.1 Microtubule/Organelle Motility Assays8 h1 Y# B h2 Z
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin4 e% g: K. @8 V8 F9 v
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
" w! K2 V, U E2 R: X+ L# \2 ^5. Unit 13.4 Movement of Nuclei
7 K% F% l |* ]; p! s0 w0 n" c+ |6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching5 v1 L' s. n" F4 o
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
, e" s6 o" a& G) a! J0 q( Q16. Chapter 14 Signal Transduction: Protein Phosphorylation
& W) Q4 t: P4 Q- I7 O% s1. Introduction) Z% j4 G& u# l& m( Y' m* \; U
2. Unit 14.1 Overview of Protein Phosphorylation
2 @+ u& }* F1 W ?" h& h3. Unit 14.2 Immunological Detection of Phosphorylation
" Q7 H9 G1 ^" u. m4 \' y n5 ~* G( G4. Unit 14.3 The Detection of MAPK Signaling6 S ^3 M: z6 U0 R
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation; C4 @% q. O5 Q/ E/ T5 d$ ?
6. Unit 14.5 Phosphoamino Acid Analysis
& V: Z! {* g- a) K5 Y# r2 R7. Unit 14.6 Determination of Akt/PKB Signaling3 \2 n; Q% u4 P* {
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
4 E, k* Q9 L! u9 h( ?9. Unit 14.8 Rho GTPase Activation Assays( T T& Q( |9 G; T
10. Unit 14.9 In Vitro GEF and GAP Assays
. a9 M9 L: Y$ _' K" Z6 o11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance+ D2 B* y, }8 s2 [! x
Energy Transfer (FRET)
+ t9 U, e, I; a& j0 J12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells1 @: a+ [/ Z* I' ]
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells& H9 y) f" s3 }0 A6 i# J) b! p
17. Chapter 15 Protein Trafficking
* v7 J! }$ A; Y4 N& E1. Introduction
, H; z) p8 G9 h. N2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways4 l- D" `5 W& N1 C" U' K
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking7 ?7 _/ b1 y! R/ T
4. Unit 15.3 Endocytosis: Biochemical Analyses
) ~$ r1 u& ~% Z0 O5. Unit 15.4 Determining Protein Transport to the Plasma Membrane. B! \% ` h3 D y' w+ K+ {* Z4 O
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
8 e6 I: P+ \, I! Y7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
; I6 Q7 H* G* w3 b5 Z8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation9 ]+ ~% p1 j+ D; N3 x6 F
9. Unit 15.8 Analysis of Protein Transport to Lysosomes: b- e" Z4 a! C
10. Unit 15.9 Studies of the Ubiquitin Proteasome System0 e0 F* Y3 o. S3 f9 B: {
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
7 h0 T, L" Z1 @1 f. `12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
, r$ A3 t/ D% r- Z13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells3 K2 @- j) \8 h( W* o# i6 [
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging3 f* b: l G. g* ~: H$ u% n: |' \! m
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and; c1 j' B6 k/ X$ A: `6 p8 ?
EGF Receptor9 k) J, I# b- u3 {
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers4 d" N) X! P9 F1 J5 b
18. Chapter 16 Antibodies as Cell Biological Tools
) y1 J; Q; J+ f+ t, z1. Introduction9 ^" U# y( r( k5 N8 e4 y
2. Unit 16.1 Production of Monoclonal Antibodies
w0 u4 ~% T, u2 J3. Unit 16.2 Production of Polyclonal Antisera
3 n0 D7 P" }* G6 \# a) B" C4. Unit 16.3 Purification of Immunoglobulin G* X9 Q& R- o/ E0 p# N: E" z
5. Unit 16.4 Fragmentation of Immunoglobulin G1 ?( w) x& i. R
6. Unit 16.5 Antibody Conjugates for Cell Biology
$ P. M( }! k/ E4 y! e7 E: M" G- A1 N7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
5 s( x1 n; N# L19. Chapter 17 Macromolecular Interactions in Cells) ]0 O% c( r p! j0 X
1. Introduction% }) ~" B0 F/ p* j+ G Q9 P
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
; h) ?0 C+ m2 s6 e+ tMicroscopy. M' V' a- E2 P5 K) [
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis' ?6 X* ?$ K/ K: W3 Q
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
6 x( B. S$ ]0 |% Z# ^; k5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries0 I% P8 X0 I" X& n3 D" h0 C4 |/ w
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry* Y9 j ^1 i' x* U: h
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
z$ b7 @$ _! o6 I8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
2 K1 J: t* a8 M/ G% C- T+ \9 LGenomic Sequences In Vivo
T( R7 i& k/ b7 O1 [9 W. q- d9. Unit 17.8 Isothermal Titration Calorimetry
2 o2 J- X! @1 g: e/ j3 M: b10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
$ Q; M. J3 o% q4 V11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
5 y# N# f# j+ b; c12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein5 S# S% u4 A# c4 l$ R g% {
Interactions (RFAP) in Live Bacterial Cells7 f+ u1 `3 V1 j& o
20. Chapter 18 Cellular Aging and Death' u$ E E# N5 R, c2 L2 n" q. o; T
1. Introduction: E: x+ P. }( H L# v5 C
2. Unit 18.1 Current Concepts in Cell Death8 L* e( W# E# R
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
) P# L1 i' t1 r* C- F/ Q4 x4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
4 F2 S) \) T5 R, U Y [6 k- t5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
0 k6 l( C2 c- `( n: p$ M& B5 @+ N7 J) w6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death8 t$ q$ u& c# o
7. Unit 18.6 Analysis of Telomeres and Telomerase& B* D) Z. c( W0 x6 p: K
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
; ?/ h6 I) ?& KPoly(ADP-Ribose) Polymerase( A1 H- J5 f1 n4 Z( c7 ?( }
9. Unit 18.8 Flow Cytometry of Apoptosis! }" x1 M! s* x M. n' w, x4 M
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
# a6 M2 G ^0 u& Z& VAssay ?( s; R5 T* z! H8 x4 w2 T7 `1 M- }
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis3 Q+ F: k; }% i$ X. q& a
21. Chapter 19 Whole Organism and Tissue Analysis
8 P `2 c( I* c3 ^* e3 x( S1. Introduction7 G6 w' i- Q+ h) S& q
2. Unit 19.1 Overview of Metastasis Assays
* d/ a; A, G' [& p+ l( U* Q) K3. Unit 19.2 Tail Vein Assay of Cancer Metastasis7 \7 R0 D( z A& A
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene: C8 K- o+ s# P$ I
Expression After High-Fidelity T7-Based RNA Amplification
8 U, S+ ?, r: } u( D5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
0 Q. ~: x( d% N1 A, M" p" q6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model/ V ^, ~8 R' y7 e/ L
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
% B5 }6 D D, \+ L) [' s3 H A8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
3 y& L# o' _' S, l0 W( ]9. Unit 19.8 Embryonic Organ Culture" {3 S* M& c1 j
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
3 K+ T' x* S. |: Q# r11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice! M" G( s) p' M2 l0 ]4 b
12. Unit 19.11 Generation of Transgenic Mice
4 x4 x( v" I S7 L7 p13. Unit 19.12 Overview: Generation of Gene Knockout Mice0 ^ I+ C7 x- F
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production: j5 L) O+ R) p5 ~! d/ w
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection& d: r" I6 e# y( N: N4 {9 ~! w
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
/ H# T' I- U4 y8 M% o1. Introduction0 K8 z1 R; Y; p; T, P8 L& m, G
2. Unit 20.1 Direct Introduction of Molecules into Cells
0 u8 F5 U5 i3 ^8 ^& Z3 W5 _" G- g- [3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System. U5 k. a1 w X7 S$ A$ M
4. Unit 20.3 Calcium Phosphate Transfection
. K: R( s8 r4 R5 H5. Unit 20.4 Transfection Using DEAE-Dextran
/ [/ i; A. U; u" s2 x- C# z6. Unit 20.5 Transfection by Electroporation
! E7 }! W8 d8 C2 ?6 s- @7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents7 B6 O- y' [0 E0 o
8. Unit 20.7 Optimization of Transfection
( T" E6 @ y& j9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
U! c& O; R9 q9 F' z1 B! a" C9 I23. Chapter 21 Fluorescent Protein Technology |: j% ~6 J. |9 Q' l. O
1. Introduction: X5 y! @0 g+ F9 N
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
1 `; _! m5 T$ z+ ~8 x3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
) q9 g3 K: }, u% w) i4 w4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence7 B7 {/ Q8 O/ M
Complementation (BiFC) Analysis
1 j' o* ~$ J: @( K, s( K5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology7 o. |$ G }8 o% g7 D
6. Unit 21.5 The Fluorescent Protein Color Palette
^( f, s! T) B' H7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
$ V4 S# U( l; H24. Chapter 22 Cell Biology of Chromosomes and Nuclei
2 \& }! |9 s ]$ C1. Introduction
E6 }6 y. U' e# l2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
& p7 F, o% D0 H4 H# Q9 `3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples. Z/ I. d+ R: }- D
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis a$ d, F9 O0 g, ]" X3 ^4 K" F1 i, X8 c$ O
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)( ^+ Q0 `$ k, e' M$ T2 G
6. Unit 22.5 Multi-Color FISH Techniques. R C9 O0 X1 Q- a
7. Unit 22.6 Comparative Genomic Hybridization
" }" ^9 ?8 Y& H8. Unit 22.7 Sister Chromatid Exchange+ b6 E' L1 {" \# T1 K+ l
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery: p8 \6 f! P7 v2 a( b
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
! m5 r% h6 s @11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs8 z! E: H& Q7 {' L8 u& K
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly, }: y4 G9 f: D
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells" U0 H7 \2 t u. W# ?" w
14. Unit 22.13 Monitoring mRNA Export
; G4 d0 Z# d( ^& X6 d15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-& B- S7 ^* F$ \0 R4 a
Field Gel Electrophoresis( C% k# X/ Z5 G0 F
25. Chapter 23 Stem Cells. J7 G- D3 ^* n$ M1 T c
1. Introduction% D# _$ j; h, |! X7 w6 l# \* V
2. Unit 23.1 Stem Cells: An Overview% G7 \9 h$ Z! K: p$ F2 P. G5 `! m
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
% |4 q) s$ |+ _6 Gand Differentiation as Embryoid Bodies
4 {& d! Q3 ^ X o4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood2 a M" V' S4 Q" G
Vessels! m+ V7 F) a0 v/ }* x1 m
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
& T. X9 T$ o5 E% \6 D" w8 f; lAdipocytes8 e7 A* K7 G; S. T4 R2 I5 Y C
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
: Q6 Q+ G) R& H1 I4 d7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells# z) D1 s2 X* N# C# N, G
8. Unit 23.7 Neural Differentiation of Human ES Cells7 J, r5 [3 ~* b' I5 T: i* M
26. Chapter 24 Lipids
9 j. ?; T# N4 |; G1 R/ r' Q1. Introduction
; |! E+ \2 B5 M* n, _7 Q2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
7 m& F2 |( H* {4 w- m8 L; @' F4 |3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
) U) r5 i4 K) P% m2 m" X: H, q3 X4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film( y# {+ ~3 z* m
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
+ r i j0 a; B7 B0 ^27. Chapter 25 Nanotechnology; b; P u% p) O% ~" \4 E
1. Introduction
( J: `' [! y# d" i2 u0 `2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
+ [* G1 L; I) v9 t/ a' ^% H3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering: h! u4 ^: J7 U6 W
28. Chapter 26 Viruses
! ], i' W/ n( M. P1. Introduction( s. g1 Z8 i8 K6 P! R0 Z
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors8 I% ~" H* F( P
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
' N7 j( S) g' @1 M( F- V$ _Cell Entry. R. a6 W y; J# Z- I" u4 t
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
1 f+ E Y" h& ]$ i7 P5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
0 _5 N+ l" x0 {* [4 Y7 f5 k6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
! C* S/ k' w9 T- @; k/ t29. Chapter 26 Lipids, [: g0 q0 ~) K \7 t- K
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by8 z# A9 R$ L% ^; U, p2 Q
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
+ [# x" e- ^$ L1 D" A2 q O30. Chapter 27 RNA-Based Methods in Cell Biology+ P5 p& V1 R" H1 n& G: z* `+ f
1. Introduction
5 D! s. T- _! D% R2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
. w: | G3 o: r: r3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
6 Q5 @3 _* w' j! s8 b. |Rescue in Mammalian Cells
+ M7 q3 S! Q5 k31. Appendix 1 Useful Information and Data& q2 e: K) i9 i0 ?& U1 K* s0 V0 Q5 _9 Z
1. 1A Useful Measurements and Data% X# q, n- Q; f7 K& _
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
. `7 t, ?9 a0 U3. 1C Identification of Motifs in Protein Sequences( T0 e$ c: g9 R: Z% I6 J0 H
4. 1D Safe Use of Radioisotopes4 \5 z2 ? e# F+ q$ d, l3 r
5. 1E Absorption and Emission Maxima for Common Fluorophores
& \3 L/ q: E1 _+ ?( C6. 1F Importing Biological Materials6 x2 V& ^) x- s. Q: T; L
7. 1G Centrifuges and Rotors
4 Q: @3 h! d+ r7 B8. 1H Internet Basics for Biologists
- B. X4 G2 M! b2 z5 H32. Appendix 2 Laboratory Stock Solutions and Equipment" E2 _2 `1 H8 K9 j7 O
1. 2A Common Stock Solutions, Buffers, and Media
+ A* p4 E1 S, M: s! T2. 2B Medium Formulations
* N7 f- j7 O" ], Q% a/ n. b5 C3. 2C Standard Laboratory Equipment J# J- d8 t5 m0 q- K
33. Appendix 3 Commonly Used Techniques
. a1 g2 x. |9 U& X9 O% G1. 3A Molecular Biology Techniques8 A# G v# x. T* k# V9 l- p
2. 3B Spectrophotometric Determination of Protein Concentration
8 C( L9 `, H6 h$ B* Y3. 3C Dialysis and Concentration of Protein Solutions* o0 Q6 B! W$ A! ~- z
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy; _& b5 x8 }- S2 s" H
5. 3E Silanizing Glassware1 [# v$ @9 o4 n6 i3 X$ g; k
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization! y9 S/ P1 X* p7 m8 k5 u: N: H% ]9 S
7. 3G Micro RT-PCR
d: `* l( a1 }6 L8. 3H The Colorimetric Detection and Quantitation of Total Protein5 k& C! P7 d, @6 |- D h
34. Appendix Suppliers# ~) a, l3 X) _
1. Selected Suppliers of Reagents and Equipment. O2 Q" ~4 U4 F, N/ X5 a6 \
* j L4 V" r- k5 q0 }" F1 S
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发表于 2011-3-8 15:53
