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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 ) f5 j/ I% Q2 z9 [. g* y
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Current Protocols in Cell Biology 2010年完整版 5483页5 d& B; x& e) T& q+ x
! E0 n" z! g, o4 UOnline ISBN: 9780471143031
( U- G: {$ M4 }6 m$ N8 s* g$ N [DOI: 10.1002/04711430309 C% H' m! F% _) ]2 c) Z8 i
5 ]* n& x: S1 {! ]4 u/ ^! G3 l$ sTable of Contents. K& C+ D: V% b/ S; [
1. Preface4 E# g# `( r! X: L7 N
2. Foreword
4 o$ F% [( W" |1 q0 Y; J0 i3. Chapter 1 Cell Culture& R0 B+ r; ?$ {/ S6 {! `0 X
1. Introduction* x# k( i3 @- S6 k" ^' _' V6 M
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture; @" \# g# b9 K6 v2 Z& O% O4 L
3. Unit 1.2 Media for Culture of Mammalian Cells
% \7 m5 ]; E7 ^( Z5 p( P4. Unit 1.3 Aseptic Technique for Cell Culture
4 Y. j3 t" w2 @1 E* g8 M5 r7 [/ L7 y5. Unit 1.4 Sterilization and Filtration3 h. G, Z" X9 h
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures7 {+ p8 g( g# P+ E* ^
7. Unit 1.6 Media and Culture of Yeast2 c' h* a2 p& M1 J. O) r
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
1 Y# X6 C8 ^( |/ y- J! R4. Chapter 2 Preparation and Isolation of Cells- `+ G6 s/ C: c' o4 g
1. Introduction3 |2 q7 b+ }% y" x5 M, K) \
2. Unit 2.1 Establishment of Fibroblast Cultures8 N1 Z p+ [; U- X0 ]% f
3. Unit 2.2 Preparation and Culture of Human Lymphocytes+ ?6 l4 H+ e" m" `' I
4. Unit 2.3 Preparation of Endothelial Cells1 Y2 i" l! a) I. }# t# i
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation) Q" S" @: r# _7 y( a8 N% B
6. Unit 2.5 Laser Capture Microdissection% A0 `% s# P; U
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures% d5 h% g; j7 Q' \+ a+ \
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
' B( n/ {& u( k0 G6 I- E+ T5. Chapter 3 Subcellular Fractionation and Isolation of Organelles8 D/ }7 ]( p9 J( ` S5 Z
1. Introduction0 {4 d* r- |! b) a
2. Introduction2 N4 V" U7 j. O- c8 N; m4 @5 r
3. Unit 3.1 Overview of Cell Fractionation1 {- @4 ]% i! A' C2 b) I" |
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
+ {" t4 ~; H; J$ M5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation# u- w! H- Y G& l0 x- N
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
" {+ c6 ?) C/ V. B7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
5 W1 k3 r; h' b5 x$ BCentrifugation2 S% b) G f/ v& _0 R
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
6 m% d+ ]1 f" @9 u* hCentrifugation
) C$ ?0 i" M1 y' s8 p/ z+ s# Q9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae7 d' i: X& p2 u! u. a$ A0 y$ `
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae; H- J F7 V" ^8 ]
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient' S- E- f7 N* m/ u- ~! k
Centrifugation; d: C6 t5 S, s* x1 _3 o6 u) C+ A
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues5 s7 d! L! C+ S$ ~# P
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes$ t/ }9 D; R+ c' U9 D' }! f
14. Unit 3.12 Isolation of Synaptic Vesicles3 P; o$ P; x, D0 n
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
; @. l2 b. K8 ^* U, n( b; i( A( a16. Unit 3.14 Isolation of Melanosomes) O6 F' }1 S4 s' T
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
+ f& K, j5 U9 y7 t: e18. Unit 3.16 Isolation of Mast Cell Granules" n% L2 t, l9 G' Y
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster) \- c$ d$ K/ G3 b+ f
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
! \3 I3 v( F8 e% q6 c6 w( ~0 s' }4 d: B21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
* l9 l9 Y1 E6 ~/ Q5 I/ t! g22. Unit 3.20 Isolation of GLUT4 Storage Vesicles8 z# ]( Y# k3 ?" F
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes4 V' P6 j( k9 i3 Y3 `
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological% Z6 W" `" R" v) j$ |& A. P' {; F
Fluids
0 {& L0 t# E3 \7 o! _2 Y$ G25. Unit 3.23 Isolation of Intermediate Filaments, h. ?- l+ D" l
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle8 r9 Z7 E$ n, D
27. Unit 3.25 Isolation of Myelin; X, e* A3 ? `. Z. ]. e# q' ~5 R
28. Unit 3.26 Isolation of Renal Brush Borders" d) v0 D5 B+ F3 q6 x3 S
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane# D# `/ k9 U( x
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
. \% @- l3 a' o9 Y30. Unit 3.28 Isolation of Amyloplasts8 `7 {$ j% Q6 G/ y9 s% A
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins8 U$ B5 |9 j8 z3 c8 }( ^! x
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
; k2 r& m- W J, sCentrifugation
. A' h! a9 E% b- V33. Unit 3.31 Isolation of Neuromelanin Granules* B" ?+ F' J+ `
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and7 k+ `% I5 O: _, i7 S
Density Gradient Centrifugation
) g; j4 L( l6 L5 j" a- a2 r3 T: }; \( M35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
1 V9 {% M5 d* ~8 p3 n36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation% }. U' |6 j {3 k" _& U
37. Unit 3.35 Isolation of Platelet Granules% G4 `+ M& \+ v3 h6 G$ u, s; t" A
38. Unit 3.36 Isolation of Nucleoli
8 W2 I! ^4 ]! l( x1 x: [$ t# V39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
a$ D( ?% @5 \40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates& X1 q9 d2 c4 K8 }2 v
41. Unit 3.39 Isolation of Chromaffin Granules
" q- @' ]. {% o8 [) w2 e1 V42. Unit 3.40 Purification of Ribosomes from Human Cell Lines2 U- a' b% k! [1 `) `9 |
6. Chapter 4 Microscopy
6 F% U% {9 C0 y' E" f% G1. Introduction. p0 [+ @. @. B7 F
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope# ~6 [* }4 |% B7 k) j
3. Unit 4.2 Fluorescence Microscopy+ x" s' N9 }) {8 V/ Q+ H
4. Unit 4.3 Immunofluorescence Staining
/ P6 z7 [! Y: x- G8 k2 `5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
4 Y- T: |( [" o; s1 Z5 P6. Unit 4.5 Basic Confocal Microscopy
) F& d" J+ |0 k/ ~8 R: s- {7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
5 S( @& k7 j9 c% ~3 j7 L' k8. Unit 4.7 Cryo-Immunogold Electron Microscopy
/ J! ~# j( [: ~) R9 I' q9. Unit 4.8 Correlative Video Light/Electron Microscopy0 J/ v& ]1 {+ a! e8 d2 a
10. Unit 4.9 Polarization Microscopy: @7 B& t3 y- V2 {# I; i% v
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells& ?6 t- L- W" ^$ F7 L& {) O; s
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues% h4 P" E) l. b
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface3 f3 @6 r5 V+ R5 V- m
Events9 S( R( g; `; T% D6 f" c% ^ |
14. Unit 4.13 Fluorescent Labeling of Yeast+ N" |) U7 f# L* |
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
* a K+ i1 A1 _, V9 U# X: v16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
: Z/ p- I! V9 e4 J& K& N# G17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules$ A. J4 Q( N5 a5 C V2 a) a" z
in Endocytosis
0 V8 C0 B2 n9 I5 H+ w, n8 z8 |18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
: [. d8 ]/ U. V' Z2 |% h% W19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development5 U3 A! |% \: w% T) y. ?7 e
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images# y v2 G0 q% H6 Z/ ]" _; j
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions5 g# h ?# ]6 X( H% ]" f7 [$ f' q
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
5 u$ S9 E( C' m4 P/ C( C23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics4 n* D+ f# P; w$ ~
24. Unit 4.23 Interference Reflection Microscopy
" s2 \; M6 X+ ]% ?25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach# X4 G9 m9 W8 G$ X$ e% |+ d
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
( u! D& @+ ~) y. u3 C# c27. Unit 4A Organelle Atlas: Appendix to Chapter 4
: E3 E0 B9 a5 b7 Q" h' Q9 b3 l7. Chapter 5 Characterization of Cellular Proteins
. t. ]% V# J0 R+ }1. Introduction
- T$ A, l/ K. T. ^1 \6 _0 b2. Unit 5.1 Overview of the Physical State of Proteins Within Cells M" U: q2 q- s; ?- q% H9 n" G
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein F: }8 ~- u. x- E6 s$ Z
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
3 E+ `3 E# C/ e5. Unit 5.4 Analysis of the Association of Proteins with Membranes- d/ f* ^' I% s" `6 K" F ? r
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)3 x; F$ {# O" K% T
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass# l" p! T( k+ ^- d; j2 M% O, |
Spectrometry
1 f' n9 i' P/ l) ?' I9 m7 j- s: `7 i8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening! x! p' l6 v) z, N" `
Applications0 s- A4 @1 J: R( G. M
8. Chapter 6 Electrophoresis and Immunoblotting& h. ~- x7 S5 j- \ T f) @1 l
1. Introduction
7 _0 i& w& J$ R) J2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins4 t( Z8 I( U( n) b4 p+ V5 w) w
3. Unit 6.2 Immunoblotting and Immunodetection3 e" j$ V4 G, L @6 |6 t
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
- i* @( Q9 Q; h2 ^+ q/ w4 u, c5. Unit 6.4 Two-Dimensional Gel Electrophoresis
1 n8 o# ?) L' U7 G& b$ l, }6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
# s* M, a* _2 @7. Unit 6.6 Staining Proteins in Gels
. n6 x: i$ y( t% {8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
5 k5 I; {! a8 G9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots6 Z& T/ \6 ?6 n. ?6 z
10. Unit 6.9 Digital Electrophoresis Analysis" B) K4 E* X! D' y
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis- r. l; w+ q, V! R. l% G2 i
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
0 s& k8 {- `3 ]Single Cell Gel Electrophoresis (Comet Assay)
% W" K: e/ o R9 E+ o# a H9. Chapter 7 Protein Labeling and Immunoprecipitation
0 U# B% t8 G$ @+ S4 j# q2 U7 |1. Introduction
5 e5 M* s; N c- S7 G/ ^/ c; E2. Unit 7.1 Metabolic Labeling with Amino Acids
( _$ c8 w) }$ Y) _ O6 P( C3. Unit 7.2 Immunoprecipitation" y( J0 ^' X1 Y" B* M
4. Unit 7.3 Metabolic Labeling with Sulfate+ ?0 L% i) D) P) Q6 w
5. Unit 7.4 Metabolic Labeling with Fatty Acids$ v! {) e8 H* b4 v
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
/ z5 n& N! W5 y" o7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins% I$ U, s) k; E
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins+ D9 c8 z( X: q7 G
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
# v6 s/ ^3 H" N, {0 {10. Unit 7.9 Analysis of Oxidative Modification of Proteins7 r6 H; b6 d; I, Q" H
11. Unit 7.10 Radioiodination of Cellular Proteins; H# T- q+ [" s# r' w' L' g3 W
10. Chapter 8 Cell Cycle Analysis* C5 y; |7 [- T; j6 V
1. Introduction
: i+ C: \5 z: q2. Unit 8.1 Overview of the Cell Cycle7 ]+ O# z2 I, l% ~2 v
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
" ~. g3 k% `, U5 P J% `2 _$ `4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle- R+ T# k. ?% a" U6 p! e
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry6 a) T0 }5 R9 |/ p5 i: `% U9 r
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells, k5 N# r$ t! f. [" I0 k
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
$ J0 F& L1 M- e+ k11. Chapter 9 Cell Adhesion4 Z0 O3 v) b" H- x
1. Introduction
! C n* h5 ?- K! Y" j. Y6 J. J5 ]2. Unit 9.1 Cell-Substrate Adhesion Assays( l" ~ r8 ]& D ~8 m+ ]* b( e6 \1 P
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
; N8 ~: E) Y! m* C8 u4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion/ C2 D5 H% g8 O ~: C5 W0 |
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion0 s4 E1 {' X( W
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules4 T/ q6 g1 [4 J8 Z& t
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions0 o' C0 n6 P+ q/ ~% b
12. Chapter 10 Extracellular Matrix$ ~' i5 q, @; V9 M# r: ~3 @$ z
1. Introduction
% d7 E9 Z6 q# M, z4 d" L2 l5 D$ d2. Unit 10.1 Overview of Extracellular Matrix
) a! l8 `1 _% b3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
$ P5 @" ^. U9 d8 a: Z5 N4. Unit 10.3 Preparation of Gelled Substrates6 M" o' D7 J' O' j @; g) \5 Z( t
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR93 v" l& w3 ?* Z2 z9 N3 w; J# T
Endodermal Cells; ^2 B) ~* x; X# f) h) U
6. Unit 10.5 Purification of Fibronectin
2 i* E6 D" @! |2 F7. Unit 10.6 Purification of Vitronectin
6 d' `$ P; ?9 M1 H- O# ~7 b8. Unit 10.7 Proteoglycan Isolation and Analysis
# \: p+ V7 `1 A, ~# P9. Unit 10.8 Matrix Metalloproteinases6 b+ D2 D! c: b7 T
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
2 a. n- \& r/ T$ `11. Unit 10.10 Purification and Analysis of Thrombospondin-1) c+ w1 N/ j3 ?
12. Unit 10.11 Purification of SPARC/Osteonectin9 g8 n4 r, ?7 t! P
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
* v* Y0 U% @2 h5 s2 l14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
5 a% F6 v8 ]4 q, x. m15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
. U2 p9 t l1 J' sXenografts% x: M, k- o) B& _
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning: ~, ^0 B/ @, F/ ]: }
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
3 h& ?! a# k' t: V6 g" o18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
7 i. ^. P9 T% l1 k3 c* ]Cell Migration
. @/ b' e8 B. [6 {7 e19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix5 x& P4 ?2 `6 _. k) d
13. Chapter 11 In Vitro Reconstitution b: y, ]( I5 t8 j: N
1. Introduction
* g( a# M) a/ j }' }; W6 s1 @2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems/ L5 {/ z) Z4 n* V
3. Unit 11.2 In Vitro Translation! j* t1 }3 g) }2 z Z( |$ Z
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells2 W; Z% W$ C5 J+ S0 z/ ?( G# V7 g
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes+ [' }5 D5 z9 N. i6 e
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
4 t( q8 y. r( Z3 v- K: ^2 ]; f) M7. Unit 11.6 In Vitro Transcription
) U3 G. ^ f+ \- K0 a4 N( ~5 X8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells5 S5 D4 W9 C' \0 B
9. Unit 11.8 In Vitro Translation Using HeLa Extract
. @1 Z7 r F! b4 b t# Q( U10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems* P3 o% `9 K4 ]* |! m/ ^1 l( l
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts j4 u' m* t; [/ Y3 m9 p
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts5 B* ^# p4 b, g+ _
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts& U/ E& T/ d" G7 z) B4 c! g
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
8 a8 v7 V! _5 ^0 a' ]15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
# ?# y4 `+ A7 ^: z) T6 s16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
t3 X0 n5 M& k( Q- n5 E; }$ B17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import+ W" ?( |( @6 O$ a2 H& k
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts1 |% ^* [( V9 U$ m
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
/ V7 R. N8 g6 S5 q0 I T% `2 `20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
1 Q3 A0 P9 J* @. y% `* D14. Chapter 12 Cell Motility% F- o- W7 ~1 l5 e6 v; R
1. Introduction' t. {" R$ n5 }" T
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells5 ]* X) M. b' A; t5 I
3. Unit 12.2 Invasion Assays
# L" N2 `$ {( y+ i4. Unit 12.3 Cell Traction
- p# T7 G3 ~; Q& {5. Unit 12.4 Cell Wound Assays" X% w; E! d' b' i9 R3 P$ u4 c5 c
6. Unit 12.5 Dictyostelium Cell Dynamics- l0 h. l1 {$ c5 d3 W4 F% m
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
C" R4 m' j* W! A8. Unit 12.7 Actin-Based Motility Assay) X3 A# N- O# o
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
- g2 A4 r8 O0 T6 j15. Chapter 13 Organelle Motility
Y5 P. [1 l, N4 M$ I1. Introduction/ p+ v6 @0 q' e
2. Unit 13.1 Microtubule/Organelle Motility Assays
u5 g- }. K+ ?* b$ m/ R' U3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
) A/ C# g) a; R `: i d4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
6 _ u8 S6 b2 g8 y5. Unit 13.4 Movement of Nuclei, g7 l5 Y9 ?7 z5 H2 }
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
9 d0 B' y+ Q( s$ O7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
8 \0 @' w1 C1 p& F16. Chapter 14 Signal Transduction: Protein Phosphorylation S. S" b; \$ o+ v
1. Introduction2 j* U( Y+ D& q. Y \: ?9 T
2. Unit 14.1 Overview of Protein Phosphorylation' O) N3 U% q g. q3 e
3. Unit 14.2 Immunological Detection of Phosphorylation# }( ~! ?6 o) X% s
4. Unit 14.3 The Detection of MAPK Signaling$ v% H# l: Y% y. @6 E
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation; P1 V( S5 `' r- }0 h8 p+ n
6. Unit 14.5 Phosphoamino Acid Analysis
7 x6 ]( _8 B n6 q8 V$ c2 v7. Unit 14.6 Determination of Akt/PKB Signaling( H4 h0 a- O6 e4 O+ Y* a4 i
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
0 W0 w7 l7 u. {) ~' ]% b9. Unit 14.8 Rho GTPase Activation Assays
5 A* T* [" B/ g10. Unit 14.9 In Vitro GEF and GAP Assays
* q. L" O% N7 X4 O11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance, t' Q( d7 V( T. x1 z
Energy Transfer (FRET)
( P+ v3 L9 G, o1 i6 E12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
6 k# ^3 n2 j3 y l& R13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
0 z0 m4 Z; A, t6 O9 _17. Chapter 15 Protein Trafficking
$ N. A6 {2 @: N1. Introduction7 n* a1 s" a. R5 T$ D9 P
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways# }! J `0 Z) B/ D- T
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking. V/ Y* ]; G7 A5 d E% N
4. Unit 15.3 Endocytosis: Biochemical Analyses% F! i' D! ]3 f4 v$ Y2 V
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
1 E/ ^ b6 u& T. P6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells! f) g- s$ y! Z4 |+ s
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum! i( h3 ~" D' q( m: y) }" p
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
1 ]2 K5 q4 D5 |1 }6 p9. Unit 15.8 Analysis of Protein Transport to Lysosomes5 b6 u: b1 z* ^( D c7 {
10. Unit 15.9 Studies of the Ubiquitin Proteasome System" f! K$ {; Y0 B4 ?$ P: @
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network4 u5 f& ^$ s- V5 b
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules& x- `' s- U/ v: _9 ]6 j4 u
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells7 y: ^; v& I+ t6 B- O( S/ w$ |! K
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
9 O0 ^9 T; @% T" ^15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
E" K/ D e: A0 F7 Z NEGF Receptor
1 B3 f4 s7 |% f" o' N/ D# E p16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
7 [% m2 O9 L! C& E18. Chapter 16 Antibodies as Cell Biological Tools
: T" A# T# D; ~9 y8 A& a) y1. Introduction' B3 K9 \0 |2 D" j1 e# o
2. Unit 16.1 Production of Monoclonal Antibodies# ~; K) [0 m! Z; f$ f% Y
3. Unit 16.2 Production of Polyclonal Antisera9 T6 Q% k! O8 N4 u5 @
4. Unit 16.3 Purification of Immunoglobulin G
! o% J. {0 M' H, M5 K5. Unit 16.4 Fragmentation of Immunoglobulin G: q% M5 K {# {4 l& c
6. Unit 16.5 Antibody Conjugates for Cell Biology
1 g: l) V1 [2 X5 g$ O7 D/ F7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
2 O3 m/ l: X$ t3 U, H* Z19. Chapter 17 Macromolecular Interactions in Cells
1 {9 @6 g2 W! I8 U* I' Q& d1. Introduction
3 |. U+ S2 O1 j7 a0 R4 i* s2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
1 {5 t2 X2 y1 m! ^3 ^( A7 U4 U" rMicroscopy% z) s; n4 y" g
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis( ]9 h9 O% P K' u
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
6 ]- h6 v' c1 z% e5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
2 Y9 D' U3 F: k+ Y6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
6 A V) O; M6 t7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors1 y1 H+ B" _+ Y; N" U' q& F
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific9 K! z: I+ v' u7 a+ x
Genomic Sequences In Vivo& _4 m% B6 r! T+ x' G) O+ x! N# M
9. Unit 17.8 Isothermal Titration Calorimetry
* \$ T2 o1 B, N7 M/ W4 s6 d& Y10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells/ f4 Z y- M) P) D) Y
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking+ D* V- |* d5 z
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
3 j' y1 \* w( h# N. p! k8 wInteractions (RFAP) in Live Bacterial Cells3 d3 C& p7 @. V' H
20. Chapter 18 Cellular Aging and Death
/ H: ~6 D' v1 T% V1. Introduction1 H: K$ y1 f: f$ a/ F' D
2. Unit 18.1 Current Concepts in Cell Death
% B" V- X: t P- A! G2 Q' s7 X3. Unit 18.2 Analysis of Caspase Activation During Apoptosis+ ]1 N- \# E8 X( ?% ]6 Y" `
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
5 b6 G0 b0 q; n" e4 L5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
2 ]9 `( b* Y- x" D! `: }6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death' x/ i$ J( {0 |1 f
7. Unit 18.6 Analysis of Telomeres and Telomerase! C5 B1 }' D6 N
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of" t; E3 G$ r2 ~; n7 M9 v3 ~
Poly(ADP-Ribose) Polymerase/ Q9 x! M' y; F5 L& E! \
9. Unit 18.8 Flow Cytometry of Apoptosis
+ K- U+ h# u% Z. ?6 W10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase. P6 T0 s; ^/ m# b% a" l1 O/ Z
Assay- x# s" ^: ^" H8 m7 T
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis& `; l6 x9 \: }# x) f& ~' A' M# \
21. Chapter 19 Whole Organism and Tissue Analysis% b; |# L4 d5 k0 y
1. Introduction$ m& n- v$ j$ S$ P5 ?9 P
2. Unit 19.1 Overview of Metastasis Assays+ q% x- k( P- ]: s% }
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
) d _3 z } P4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene5 l( ~, a2 B5 S1 h/ F
Expression After High-Fidelity T7-Based RNA Amplification
, _* i8 q+ Q4 ^2 I7 B$ `5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA( n- l$ K) l* j7 C/ E
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model% t6 t' r+ E6 h9 a7 V m. j
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo" Z( Z' y; B- K( s7 }
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
3 g# a9 D. j: s; y( W, V- `4 u8 B% ]9. Unit 19.8 Embryonic Organ Culture0 d+ F( }( `! U. q: V8 V5 X
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
- n! {, K! r$ f, c- y" D/ n6 U. c1 k11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice6 b1 r+ @2 e# v8 N" W- _! w! u, r
12. Unit 19.11 Generation of Transgenic Mice
: j" l5 |5 w8 V& `, k& B13. Unit 19.12 Overview: Generation of Gene Knockout Mice: D6 e' @, }" k- y% t
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
" [3 x& h7 ~/ q" a15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection$ y2 y+ l- x0 C; l
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
+ c; ~! V/ _- p/ V$ z/ Y1. Introduction
: D7 L" Q% w% W; P! [2. Unit 20.1 Direct Introduction of Molecules into Cells
+ e) x2 `( @3 j; V3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
+ z* j2 ]4 |* B& P6 P j1 W# I7 I4. Unit 20.3 Calcium Phosphate Transfection
5 C3 h* `! I5 D$ H- Y V5. Unit 20.4 Transfection Using DEAE-Dextran
- S: I& u+ a& f, q0 K' U. D# R' G6. Unit 20.5 Transfection by Electroporation' U7 Y" v3 r" b5 y
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents) b$ a8 v. O2 X
8. Unit 20.7 Optimization of Transfection
# Y0 K8 H3 i- J2 X7 w9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
# |, Q( o" B! c! t7 N9 m2 J- k5 X23. Chapter 21 Fluorescent Protein Technology/ T8 @: k1 ~5 ^. H9 K: B: Q
1. Introduction
* f1 o/ y% Z; Y+ H1 _2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
Y# k% t$ |' g! B3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
4 I( W; i6 x8 G4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
/ C* e( R% s: \% L4 @3 c9 pComplementation (BiFC) Analysis- _% R9 Q# b$ s& V2 |# k
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology$ G# O$ \& p1 ~2 o* j
6. Unit 21.5 The Fluorescent Protein Color Palette
5 E/ ^( N& i! ~+ v d* h7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins$ h- ^# l7 ^/ P' N
24. Chapter 22 Cell Biology of Chromosomes and Nuclei
5 n7 V# h e* k% S4 A9 T3 M1. Introduction
; t! T8 M- P- E3 I2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis7 s8 h& L% `' Q& u ]. C
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
1 _5 _% U0 N" C% t K2 n4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
0 \; G2 t6 Z) I1 s# F8 h5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
1 O4 Q' P( U; d, f9 Z; \' W6. Unit 22.5 Multi-Color FISH Techniques4 @8 b9 f8 P4 _# q
7. Unit 22.6 Comparative Genomic Hybridization2 v, F! B8 A2 v# @+ y: W3 Z2 `
8. Unit 22.7 Sister Chromatid Exchange
0 \9 y. w. K& Q6 z. y9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
7 n$ Z6 A n9 I. p. v10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
/ _5 x8 `5 G* V7 s0 G11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
2 b. z8 K% p c5 H: j12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
' `( }' A, H9 w7 ]9 W! z13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells. S [5 S" B* _. t
14. Unit 22.13 Monitoring mRNA Export
8 ~6 u7 r1 T; x$ L) V15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-$ Q) N5 ]$ B$ d( I
Field Gel Electrophoresis4 E+ G0 k: T; a! b( R. \, t( P
25. Chapter 23 Stem Cells
" p/ ? I& M/ g! D1. Introduction
; [$ ^3 r+ D4 ^ n9 L1 v, y$ ]2. Unit 23.1 Stem Cells: An Overview! z/ }- c# x8 W& g8 Y
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
% _9 A" L9 W% V' Uand Differentiation as Embryoid Bodies
, W; F, H3 j3 u% ~2 f4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood% G _+ o! P7 w2 A2 v* j" v2 L, X
Vessels7 P! h: h# i0 |1 ^/ E% ^" M
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
3 s# T. L5 K" e0 Q, I: hAdipocytes
# H4 _% D% ]7 y- J5 q8 o5 Y& ]6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation. { }) {* [6 S Y7 N
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
# e7 a, l: R/ L3 e; J8. Unit 23.7 Neural Differentiation of Human ES Cells
& Q! ~2 s0 O. }3 O; ~% i! d4 S26. Chapter 24 Lipids) Y5 D' t; W$ o+ o
1. Introduction
# @ U5 C* [$ v& |+ J& y2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking" }$ s0 j3 z! G) R9 M
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
* e( f& |8 I7 F( e, [: |" Z k! B7 I4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film+ D# ]$ u9 B! q! w, i9 I' Q6 m
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
4 f; X2 [2 w( @27. Chapter 25 Nanotechnology4 ^+ M# D! I" D
1. Introduction" p2 Q5 k4 j$ L5 e
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
% i) x+ O' ~ Q3 Q3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
; m7 X! g( E& C7 l28. Chapter 26 Viruses* R: |# F' ~# x5 v" S
1. Introduction
+ k: F& e4 y/ y7 R7 |4 X2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
|* E, {6 v( v) j9 K3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
8 X0 s2 c6 C2 }9 v+ JCell Entry: M( g; h) `% O! ~
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
7 ^# r: _. A+ i, p5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
+ j+ ]: n) P n6 d* N9 i6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission+ z5 o5 }7 I K; j) k' t% k
29. Chapter 26 Lipids$ x: T7 \2 I/ J" K
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by7 a' d- v2 w; o" Z2 p5 S
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
5 g/ i V$ `" \' [7 t8 a30. Chapter 27 RNA-Based Methods in Cell Biology7 z3 p1 J: v) Z1 J: |2 V4 e
1. Introduction
9 M0 K" [$ N* _- E. d8 {2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs% B5 a2 M$ r, T
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible2 w/ {: y% X5 F \7 ?2 q
Rescue in Mammalian Cells
) s( T* C, K/ C3 U: R31. Appendix 1 Useful Information and Data
* v Q- ]! y0 f- W8 N0 X1. 1A Useful Measurements and Data8 l h" c y# S9 D! X! \2 H- M
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research4 b i6 ?# j6 ]9 N1 r8 v+ M! F0 {
3. 1C Identification of Motifs in Protein Sequences3 b, L8 w5 f# S5 r
4. 1D Safe Use of Radioisotopes6 w) k6 Z( O& G% R5 ]" B
5. 1E Absorption and Emission Maxima for Common Fluorophores
. A) E$ j$ ^' G# W6. 1F Importing Biological Materials
% q7 T8 _9 c- R7. 1G Centrifuges and Rotors
* p. z# z1 ?0 k8 K/ B8. 1H Internet Basics for Biologists1 z9 Z' L; J2 g- j4 m- h: C* ]
32. Appendix 2 Laboratory Stock Solutions and Equipment' _/ A* l5 s- M9 m3 S
1. 2A Common Stock Solutions, Buffers, and Media
" o! d& V6 [2 c6 V! E8 i! q ^# ^2. 2B Medium Formulations6 b4 y' m% C, n3 p, \2 U- r* ?8 Y" U
3. 2C Standard Laboratory Equipment
; C# r9 }0 i! M, O, b5 U33. Appendix 3 Commonly Used Techniques
) R$ |% O; A9 W2 D+ `. Z7 r! F8 g1 F4 S1. 3A Molecular Biology Techniques1 G, A) ~4 F9 R! b
2. 3B Spectrophotometric Determination of Protein Concentration3 s3 m* M0 A& h m; \0 z7 u4 U
3. 3C Dialysis and Concentration of Protein Solutions
1 T+ V1 o0 C* F4 H' ?/ q; P- u4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy5 O, V& S! T h
5. 3E Silanizing Glassware3 I$ d: Q5 i r) ~3 p
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization9 F9 x& |8 D8 a/ f. ^$ U. n
7. 3G Micro RT-PCR5 P" J# L, H$ d8 n P- l, E# W
8. 3H The Colorimetric Detection and Quantitation of Total Protein
, D8 P0 }- v8 f/ L7 L& K6 O# X34. Appendix Suppliers2 K& Q; U! ?0 H; }2 ], F. X7 [
1. Selected Suppliers of Reagents and Equipment1 {7 H* @6 U/ `: k' A4 y" E9 M/ ]
& W/ y% @! s4 S& Z
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发表于 2011-3-8 15:53
