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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
9 c# K& F* y+ w7 f6 M O9 t/ |
* D; y. r) K. COnline ISBN: 9780471143031
' P& G; s, O) H: ^* X4 R' `4 GDOI: 10.1002/0471143030& H9 r6 |6 o! C6 z
4 H# U( ^+ Z, C; {
Table of Contents
3 G6 c2 _' r9 O. M1 A; U1. Preface
& v( Z5 ~) d& k$ R, x& b2. Foreword
& A; `! n$ @, i3. Chapter 1 Cell Culture
4 V1 z2 D4 ]0 ~1 g# p1. Introduction
5 J4 S: s- u: g$ ?; B5 a& e& R9 l( {2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture4 H8 c4 h8 }: S* D; [
3. Unit 1.2 Media for Culture of Mammalian Cells
# h, W L' n7 P* D& j& X4. Unit 1.3 Aseptic Technique for Cell Culture
: X7 u" j3 b& p- y2 S5. Unit 1.4 Sterilization and Filtration
, `) {0 m, {/ w6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
* Q9 f' w- a9 D; ?$ ]6 \" Z+ e7. Unit 1.6 Media and Culture of Yeast
1 O8 t# {7 C: i8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging& [* @5 [. h9 }1 g9 L ~4 S
4. Chapter 2 Preparation and Isolation of Cells
@- ~7 n! c! f8 o, o1. Introduction
" }& ]' C, s( n# i2. Unit 2.1 Establishment of Fibroblast Cultures
! H" ?& k$ P. I5 E3. Unit 2.2 Preparation and Culture of Human Lymphocytes
+ J! U; H. ]8 h5 @. X4. Unit 2.3 Preparation of Endothelial Cells
% K6 Y) ~: o# ?7 c+ o* A7 g1 j9 r5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
4 z6 a& B/ P2 f( u8 A9 m. t: ^6. Unit 2.5 Laser Capture Microdissection2 Q7 g9 E8 Z7 \/ h; ^/ V
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
6 p" N8 r: r2 ^3 d8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells* A! s: k0 c4 t- p5 M) N! |
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles2 m. d' f9 y$ ^& w' _
1. Introduction: D/ l# z( i* C
2. Introduction
9 X! s3 \8 ~3 D+ n( A8 n/ x6 d3. Unit 3.1 Overview of Cell Fractionation
4 \5 z" M7 X1 w/ V9 g# J4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
( A" O, v, ~3 P/ F; h* s5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
6 I, p' n7 |% A% S6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
4 }/ G' n- Q* _* T7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient% P/ U- f2 A6 l4 T+ m' @& p
Centrifugation
1 ]1 t" ^6 n. r. g9 I* y8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient9 i8 Z7 }9 | t$ h1 t7 ~7 h' f
Centrifugation, M# X/ ^" c8 L6 l$ s1 f& O7 n
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae0 L2 U2 N |7 X! `8 L0 ?( D: C
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae# s9 w% L+ U; V8 y/ B% ~* \! i- B* l
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient; ~- Y7 z M ^$ W* @' Z- R/ ?1 X0 B
Centrifugation
% i& f) k. H+ F# s+ J! m6 p* U12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues2 Q3 C- t6 z! ]/ m0 r \0 C
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes: P3 {0 P4 \4 I w. Q3 \* p
14. Unit 3.12 Isolation of Synaptic Vesicles
$ A0 f5 I1 c2 O7 P9 M% T' i15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
$ p8 H; z/ M( U3 n16. Unit 3.14 Isolation of Melanosomes$ r* U' z( a% F2 Z, R
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation3 a* k+ M& Z7 P Q2 x
18. Unit 3.16 Isolation of Mast Cell Granules
" w7 H+ U# I# t19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
0 J8 S, J4 W) M2 O3 h0 x20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas# P" k Z& s! a c
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons3 F& n0 O3 Z5 P% H0 R$ B
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles6 s/ k; {( |( v
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes! ~% E& D. `) m7 f+ _6 ^
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological+ r& k. x5 W, Q5 r
Fluids6 z! P! g3 L* Q9 U; m
25. Unit 3.23 Isolation of Intermediate Filaments
% p! U( @( Z0 t# J$ ~* A$ K26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
2 ^. } S1 ]8 b. F' k* ]27. Unit 3.25 Isolation of Myelin
4 ~* Y4 w! R e- }. [" V8 @4 d28. Unit 3.26 Isolation of Renal Brush Borders
4 v) j! p2 ^3 b; U, F* o# B29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
( Q) |/ l" z& m6 p9 RFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts0 x+ A- f+ F4 H! b
30. Unit 3.28 Isolation of Amyloplasts. x* Y" l }* \. z. N; g
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins5 G# t# x" D+ h4 [" L# j* A& G' S
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
+ S) Q6 g4 d; [8 UCentrifugation
# x( J/ E h) m4 C0 g9 t; U2 {33. Unit 3.31 Isolation of Neuromelanin Granules9 K3 j4 d, D: W0 H; ~' x
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
! G; }- f9 Y& U8 B7 i) w( _- QDensity Gradient Centrifugation9 O/ ]+ o/ }9 I* p; Q4 ]
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments8 a) g9 ]9 J4 i) C4 G
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation! L- `" E+ f2 r, H! s7 g _, x
37. Unit 3.35 Isolation of Platelet Granules5 h$ N6 [: @! E" a
38. Unit 3.36 Isolation of Nucleoli$ L3 ^; t- m$ f* @
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins/ O$ j2 _/ v7 L4 I% Z- H% u
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates u1 P R9 k" j3 @) Z' |
41. Unit 3.39 Isolation of Chromaffin Granules
3 P' o5 t' q; i42. Unit 3.40 Purification of Ribosomes from Human Cell Lines/ P. @- m. x& l9 |# ?: U/ q
6. Chapter 4 Microscopy5 S- Q7 A7 V! U) E Q
1. Introduction# a) A0 D7 v. D1 [" x- D
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
" k" v3 ~7 I% a0 P! j3. Unit 4.2 Fluorescence Microscopy
" X" t4 P. o) `( F0 k( w' i4. Unit 4.3 Immunofluorescence Staining
# `5 l1 g) F) t" E; d8 \0 d' ?5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
, c9 T5 Z$ ^( b" _* C6. Unit 4.5 Basic Confocal Microscopy2 t" W, e$ i3 t' j! r: g1 G$ b
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
7 m4 m1 [( T! K" A8. Unit 4.7 Cryo-Immunogold Electron Microscopy
9 ^$ \7 S* I9 L$ H" ?9. Unit 4.8 Correlative Video Light/Electron Microscopy
2 O! E1 l8 v* Q& T" ~1 W10. Unit 4.9 Polarization Microscopy
0 t0 E- t4 P* H/ z+ X" y4 Y' C11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells5 D- P4 o; k. i1 M) [2 a* [% E4 D, E( } n
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
4 S) f+ C5 U3 v13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
6 P m5 T0 q# JEvents4 d# F# Q0 F0 ~0 ^
14. Unit 4.13 Fluorescent Labeling of Yeast) _4 {' |1 K3 N: Z' c
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy! h) h7 s/ A* {5 X* ]
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy5 d3 v8 t5 j U: l+ p! d; O
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
8 @0 L. n) g0 Q* Lin Endocytosis1 G! e" @/ N$ f/ x0 E/ e: W
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology9 G0 M: x! \5 G: R* J
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development: v0 {- Y- G& c, z5 q& A
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
, D$ ?2 ~! {5 a. s( V% \21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions/ j: B% c+ Y: X# g0 J! K& M9 \
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
+ J, t5 a* k. j& J23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics h$ s c. d0 S3 [. [- }! ?
24. Unit 4.23 Interference Reflection Microscopy5 t) X4 _7 z/ O; R9 g/ G. c
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach) H: h$ P7 [" ?
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches2 b, j4 K0 N) h8 d
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
! t/ L' m- {: d. b1 |* g7. Chapter 5 Characterization of Cellular Proteins
4 ?+ D5 ^; f# d* {: p7 I1. Introduction
+ z- i; _, w- Z. e) Q- c# e2. Unit 5.1 Overview of the Physical State of Proteins Within Cells5 \- L/ T$ d: k' {" h
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
2 h3 v! x& t f7 ?& P* y4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients( ^7 I; V' w: w; v4 ]- D
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
1 l; j, y! q% R% `, E3 R0 C6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
* X0 K8 U3 b) R* c; Y3 t7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass8 b w2 u1 E% d! o% N! p: C! b; i! ^
Spectrometry! ~- @7 x$ x' c- \( ?; K: U' s' B- V
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
$ @3 ?8 K* {9 j3 g C/ FApplications0 S9 |; K) ?9 C u
8. Chapter 6 Electrophoresis and Immunoblotting
I( h6 s6 n4 G3 b1. Introduction
# Y% v4 [/ a+ z8 r. o2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins$ O& W" C; k0 \1 L0 \# a" @
3. Unit 6.2 Immunoblotting and Immunodetection/ N7 u7 v+ W1 i# ]4 B
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots5 x8 e( O9 z* Z. ]2 r
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
E9 {) i% H. k& o1 C; E# L6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions; }. R2 i; l" G! m+ w9 X+ m
7. Unit 6.6 Staining Proteins in Gels
( Y: e$ N! ~& S, ^& ?* N8. Unit 6.7 Agarose Gel Electrophoresis of Proteins* x" c& N g% d1 X" J- Q1 q
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
: I4 l4 w/ H" { Z- s10. Unit 6.9 Digital Electrophoresis Analysis- L( Y+ P0 u9 D* ?* w+ X
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis0 ]' ^" f5 U/ j& E8 O, _3 a7 R
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of; v2 c+ V5 j, ]" w$ i. t
Single Cell Gel Electrophoresis (Comet Assay)( _4 O5 Z: \/ |8 d1 \9 T) G" Z
9. Chapter 7 Protein Labeling and Immunoprecipitation( \( W# A0 U9 I2 n" U/ e7 l. s( n s
1. Introduction
. o; d' k6 v9 K2. Unit 7.1 Metabolic Labeling with Amino Acids3 Z {0 q+ ^: x* d9 x5 q
3. Unit 7.2 Immunoprecipitation
+ T; s6 t, X) Z5 k8 n# Y- B8 h, o4. Unit 7.3 Metabolic Labeling with Sulfate2 D' o6 p8 z# J; W) j5 u7 o
5. Unit 7.4 Metabolic Labeling with Fatty Acids3 f) a& ?. {( y% Y* g; X' |
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups# j7 E+ Y3 y3 {7 b# ~) G: {
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
& O8 X' T: l$ ~1 X/ Q7 F8 Z8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins5 X: o" m% i5 T* V5 Z0 x
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
# R e) ^) U3 ?10. Unit 7.9 Analysis of Oxidative Modification of Proteins/ z5 n3 d% e+ p4 }. r( \. o# M
11. Unit 7.10 Radioiodination of Cellular Proteins5 E# R0 p# i; {: K9 x
10. Chapter 8 Cell Cycle Analysis
0 v4 {+ T8 i% l1. Introduction
) { m6 z2 z7 k# {8 T2. Unit 8.1 Overview of the Cell Cycle
; f6 F( f4 H6 v5 i: h: L3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle4 }8 ~' g6 T% @
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
- k0 a) w# L6 c' \6 a8 |4 T* C5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
' V, A3 `' S( Q3 i- ]6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
0 T$ s# M9 m" S( N+ i6 x {* Y2 }% D: M7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry1 t# [1 n# ]! C3 C+ D
11. Chapter 9 Cell Adhesion
Z8 z1 O% E+ d1. Introduction
* c b% {* Z: ^7 `! y( I) O i2. Unit 9.1 Cell-Substrate Adhesion Assays9 `2 |. h- v4 k, h8 T- |
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
9 B! O# I% ]' I& {: ~4 z0 s- ^4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion; ]6 u0 G5 j2 S. k, B. f% P
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
8 }% V7 S+ F: C1 }6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
+ A1 D- O) ^1 @+ X! i7. Unit 9.6 Measurement of Adhesion Under Flow Conditions2 i' Z. z: n8 T% Y, k8 E
12. Chapter 10 Extracellular Matrix
( ?- s' |: Z# l* U1. Introduction
$ _& I- i7 \; w% U. q, D2. Unit 10.1 Overview of Extracellular Matrix! r+ h c( A) E- x: R! A1 I
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
0 v n& R2 h$ n ?+ s4. Unit 10.3 Preparation of Gelled Substrates
z7 F2 u. o9 a" V, H: Q2 j0 z5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR99 a, s+ @$ `8 [9 x' b
Endodermal Cells$ {# l) h& R5 b& f9 A3 o
6. Unit 10.5 Purification of Fibronectin
6 e5 y! J. u4 X; r6 l9 _& `7. Unit 10.6 Purification of Vitronectin
- Z; m6 l- }& E/ E) o- f8. Unit 10.7 Proteoglycan Isolation and Analysis
- J) i/ ^8 O4 R# ~/ \2 a9 S9. Unit 10.8 Matrix Metalloproteinases
9 u: ]7 y* f$ A) p* o, u10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts" s: k- s) ^0 B7 c) A
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
* Q8 T& G L" m, R7 t+ `: t9 u12. Unit 10.11 Purification of SPARC/Osteonectin5 Z" A: c9 {9 X8 `/ ]
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly4 y$ J4 [* {& p, {4 J8 e) k
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly$ `9 t& z8 ?! t+ ]5 x x) F
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
/ w! X5 ]" R/ |) y8 |( t$ pXenografts
- T, S2 ]" u- t% W4 [16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning5 n* [8 ~$ y# z( \
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties6 U/ Z2 C7 n/ Y
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
9 |. t+ @2 D' LCell Migration, n4 B6 ~* G, x
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix% {1 z7 }$ @! ^& \) b1 v! f1 B
13. Chapter 11 In Vitro Reconstitution' F; W5 E+ ~* {. ] j& F8 ~) C
1. Introduction5 H; p$ P, }& |# y
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems- x+ K, n0 {6 |& H" p8 u
3. Unit 11.2 In Vitro Translation
% l' ]0 _) Z6 A; X n: G4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
m/ C# t+ Y7 e" K7 y/ M5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
1 M" m0 L; ~( _6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication- \: Z8 g q8 \1 }7 J/ Y
7. Unit 11.6 In Vitro Transcription+ L+ n6 j4 \0 j) T P6 `2 n* a( D
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells7 L3 i; }! c5 M: Y) Z
9. Unit 11.8 In Vitro Translation Using HeLa Extract
* i1 e; V7 L2 R. g10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
! x. v' M0 A* ~0 ]8 z" y: j" A2 p11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
- k: r/ m; u* u7 N9 |12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts6 h# X, f2 N+ p5 k& `1 m
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts: Y& U" j" f+ q+ s# s2 O; P! O2 P
14. Unit 11.13 Mitotic Spindle Assembly In Vitro+ q! |; b% l% [4 I4 J$ O0 U
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
9 `8 s2 T# c/ u16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
4 c8 J1 G) T5 G- e# c8 G/ Z: R17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import- S- h& u* \. d& N! H9 v4 H1 G
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
$ c& l) `6 U' F) T0 a1 c0 @4 o19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
: `6 o" D# A& ~+ S5 ` i20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
: @# e' k+ q( D3 Q7 d; K14. Chapter 12 Cell Motility
. j! X4 M n5 b, m0 D( \1. Introduction8 b+ }( l. A) j$ o. E
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells7 ~; P# `, {- e- E2 k- I
3. Unit 12.2 Invasion Assays
$ f2 U& w" q# e) J* M% S! A; {4. Unit 12.3 Cell Traction
8 p3 B; y" y) J! O( Y5. Unit 12.4 Cell Wound Assays$ \- u( R O; r) \ _
6. Unit 12.5 Dictyostelium Cell Dynamics; [* p$ [( j/ i9 p' Q* U+ O
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
X4 K- a- H( D8. Unit 12.7 Actin-Based Motility Assay8 B& z3 G* _) \7 M) M% ^" {
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
c: ^' c: Y+ e5 l15. Chapter 13 Organelle Motility
3 a. q) L7 R' N+ n( R1. Introduction$ \% Q' C$ [6 R( o
2. Unit 13.1 Microtubule/Organelle Motility Assays
/ {2 o; \+ L+ W9 d. R7 Q3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
0 X8 A+ b# g) u" Y6 c4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP* q; v5 E' P3 u2 O/ b
5. Unit 13.4 Movement of Nuclei- ?. W% q! x! H, M% F, J& z
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
4 X% T1 f( O/ j7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly2 C3 D1 [7 b1 Q# v7 u
16. Chapter 14 Signal Transduction: Protein Phosphorylation
! W* C- N6 t6 K4 K0 i1. Introduction X/ p1 T0 X' B- h
2. Unit 14.1 Overview of Protein Phosphorylation
# Q0 q+ {# r7 W! j1 b: z3. Unit 14.2 Immunological Detection of Phosphorylation2 ?+ D6 v; w! p- B3 \5 W" ^7 G
4. Unit 14.3 The Detection of MAPK Signaling9 i- u$ V- F7 \! E2 D+ u
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation* ?. y2 J0 n# A* c/ B3 y4 y
6. Unit 14.5 Phosphoamino Acid Analysis: c* O$ @; o8 V% C2 b0 C& H
7. Unit 14.6 Determination of Akt/PKB Signaling! o( B9 `# |3 p
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
1 U* c' Q! y O$ y" u0 @6 c9. Unit 14.8 Rho GTPase Activation Assays q. o& z6 p6 b: v
10. Unit 14.9 In Vitro GEF and GAP Assays
' k( v+ M# X+ H11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance+ J$ K! \% y& x0 p8 F8 ]7 r
Energy Transfer (FRET) S M7 J2 f- h1 ^2 I; \
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
2 @7 d( @ I' l/ O& @7 [13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
4 ]2 t- N" z/ |" @5 Z# c9 ^( D17. Chapter 15 Protein Trafficking+ I6 |* _9 ?1 z9 g- u) f8 J2 q
1. Introduction
% E8 u& k s" L0 m2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
7 R" S7 k9 K$ F; v1 t: Z3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
8 b0 M5 z& f2 J4. Unit 15.3 Endocytosis: Biochemical Analyses
/ M& k* B4 b" T7 }1 ^6 J2 {1 W6 v5. Unit 15.4 Determining Protein Transport to the Plasma Membrane; O. W9 X; m0 _7 Q) p( K
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells! s% W( T7 j, }# ?" p2 e1 |
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
, {3 h$ ?6 [6 i' o/ O6 U, `% r8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation5 ^, }0 B$ c# N! J% c7 y$ ^
9. Unit 15.8 Analysis of Protein Transport to Lysosomes6 _( O" r: O6 ?- Q7 g( ?( |$ U
10. Unit 15.9 Studies of the Ubiquitin Proteasome System& {4 c4 [$ `: s9 G8 R
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network& R* ~7 z2 ^' I% l* j3 E+ ~; l: l
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
, _ D3 v: E( g! C" P# `7 v13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
- B3 J! o d2 C1 w- Y, T' G; p2 X: A14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
0 j! e5 N# I4 T15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
+ }' v% G+ Z2 j: ^3 QEGF Receptor
0 F$ D4 ^+ z6 ?- K16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
; F* C2 p, x+ t1 `8 ]18. Chapter 16 Antibodies as Cell Biological Tools/ v' `, Z$ d& ]: x* l% Q# R
1. Introduction7 F* W7 J: b( f
2. Unit 16.1 Production of Monoclonal Antibodies7 Y' G' ?+ X* Y, ~( Q1 g3 t
3. Unit 16.2 Production of Polyclonal Antisera L" M6 v# |4 ~' _4 ~, ~& L" {
4. Unit 16.3 Purification of Immunoglobulin G
h. P/ d3 I3 x5 C) W9 U5 T7 n3 o5. Unit 16.4 Fragmentation of Immunoglobulin G8 G6 I5 i, g$ \9 T8 k" y
6. Unit 16.5 Antibody Conjugates for Cell Biology
( E, G4 Q6 ~. e$ W' V" [7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
$ C; q: }) _5 v. i( P( v19. Chapter 17 Macromolecular Interactions in Cells
6 C! X6 U4 `: o# S( C- O) Z1. Introduction4 Q6 r) V! f! }/ l6 L; S4 A# e
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
1 B* w/ { k8 N* @# kMicroscopy i! U6 J2 U i# I
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis4 r0 t7 p) O b
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins& ]% h1 d# k: I. h( m
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries" q! Q9 W& A S3 w4 X, h
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
) o/ F% J1 m: J, n- Y3 N+ J7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
8 F6 j) k/ R7 q/ V$ [7 R7 p& |! f8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
% P- U: p) w( w# S# Z8 [3 A; ^( bGenomic Sequences In Vivo
?6 `8 |7 }, i& D8 M" M0 T# t9. Unit 17.8 Isothermal Titration Calorimetry
5 B( {% G. @5 j% K: T) i10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells3 @1 S3 I4 \% e" ^& m; C' S. X2 g5 G
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
% K; {, J$ h" N9 O7 n2 ?; D$ b12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein# k1 k# T7 e" v% b
Interactions (RFAP) in Live Bacterial Cells
1 q: B% ^8 B# L$ B. D2 z% n20. Chapter 18 Cellular Aging and Death, Q0 t! K/ b C
1. Introduction
! `2 T: c+ f) R- d! C* {. f2. Unit 18.1 Current Concepts in Cell Death
z: _' {. g" W, ]5 e3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
1 H4 t' | b- }+ Q C4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
1 r$ U; y/ k# k6 e# {- n/ q5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
$ Y) U& b# i1 ^( \- J% g# b' O3 `6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
. S# k# p7 a Z: i3 F, m+ F& v7. Unit 18.6 Analysis of Telomeres and Telomerase
2 G' S! X3 j) \6 s! \! e$ I8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
0 M% p( L* D3 F! _1 J$ ^Poly(ADP-Ribose) Polymerase1 K0 {6 k8 v* ~
9. Unit 18.8 Flow Cytometry of Apoptosis
! g9 d# D- r% N10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
$ ^) B# g$ p1 t8 xAssay
" v3 |$ s: C5 E9 y6 T11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis! H5 n& n) B' _3 U* z h* r
21. Chapter 19 Whole Organism and Tissue Analysis; C/ e9 R7 m$ c; ?1 N" }
1. Introduction
) O& \" r( I# ?* E" B) q# E2. Unit 19.1 Overview of Metastasis Assays% r5 _1 O$ M2 r( @3 v. V
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis+ \$ z V+ w4 u& e
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
; `2 u' y% P/ M# \* d0 hExpression After High-Fidelity T7-Based RNA Amplification
8 L" Q* Z5 N+ g5 B' q' Z% u5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA% Y1 B' G; S8 w- r6 Y$ `
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model" m ~5 `1 J$ y0 l/ j8 p
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo+ d1 k$ [) i; f/ w6 ?1 t, `; \
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
6 G5 G' |% c0 h/ e# r9. Unit 19.8 Embryonic Organ Culture$ P1 ^4 g9 M+ b
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
2 }3 X5 Q' j; w2 W" D2 t2 V8 }# |3 \11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
0 \' v/ X# K- ?6 f) \, l12. Unit 19.11 Generation of Transgenic Mice
; s* D3 [- \3 `1 F+ ? f0 Z$ a13. Unit 19.12 Overview: Generation of Gene Knockout Mice) h0 i, j0 B( @1 Z- u
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
+ T! b: ]/ O# n$ x* w15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection7 X" g6 s" j n% V$ y/ Y G& J) K
22. Chapter 20 Expression and Introduction of Macromolecules into Cells9 r- H& {5 @6 V5 {3 s* f
1. Introduction
7 U0 ?( U& L2 m$ L9 z! M4 _8 @/ Y2. Unit 20.1 Direct Introduction of Molecules into Cells
# m) X, H. H6 T3 h4 t3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
' X+ E8 \6 C& b8 C. H% y! i4. Unit 20.3 Calcium Phosphate Transfection
) g1 O) B2 {+ O+ ~2 c5. Unit 20.4 Transfection Using DEAE-Dextran" F+ \/ V9 }0 K0 T% E" y: F
6. Unit 20.5 Transfection by Electroporation: G p: P* e1 z. D7 K$ L! t
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
$ W. ]. F; N- F8. Unit 20.7 Optimization of Transfection
* n# [+ A" T7 s9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System7 N) F' R, {7 j
23. Chapter 21 Fluorescent Protein Technology
' A& _( ?( S) m: i1. Introduction3 j! H$ h0 \4 F7 _/ V
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells; [8 `3 A/ O( D J) ]1 Q
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
$ _/ f* {( c: f! x3 |9 P% O4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
2 Q2 B& @4 @: jComplementation (BiFC) Analysis
' M; \ s- \" T! {6 U" v. q5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
) i, t) b/ c2 s! U. j6. Unit 21.5 The Fluorescent Protein Color Palette
{% n+ s* b! l7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins# O# X6 r) U9 O7 q" I3 T8 K
24. Chapter 22 Cell Biology of Chromosomes and Nuclei$ G! s$ k5 P# X9 s
1. Introduction5 {$ D# @2 P/ w
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
, G& x) u$ k. Y, J4 {: O4 Y3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
0 W V. B( j+ p, ~( {" S5 e4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis9 ^! v' I* [1 O, [& o. f
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)$ u" t* B1 i6 e8 n" T
6. Unit 22.5 Multi-Color FISH Techniques0 q( Y; _5 w6 g2 x4 P
7. Unit 22.6 Comparative Genomic Hybridization
/ o( v r+ x# b8. Unit 22.7 Sister Chromatid Exchange
9 X1 k" t3 E" I% @) r+ b0 o& H1 s7 _9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
% b1 N, T. ~1 G7 T10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
' ~3 L, \- F$ ~1 F11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
0 ^/ x; V9 E( P3 d12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly6 z" J- Y* b) R+ L9 _" N: }2 e
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
; E+ A- a$ c' w; o* I2 I; M$ l14. Unit 22.13 Monitoring mRNA Export; L0 @9 Y( _& e
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
2 w, ?& N6 V' ~% G D- i( Y9 d; gField Gel Electrophoresis% ~% L/ |9 g' h+ v( P
25. Chapter 23 Stem Cells
$ u, D. q/ V) i3 B# J, I3 j8 T1. Introduction% D9 y+ B0 R( s9 ~/ ?
2. Unit 23.1 Stem Cells: An Overview
* t" D; ]" b. x9 b# [! @. P2 B3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture0 r: D0 _. |0 ^ _9 Q- @
and Differentiation as Embryoid Bodies. _( ^' |3 Y O
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
" M6 i7 l& ~' w4 \Vessels! @4 `; U# `/ s- C o1 h
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
1 y2 {. I+ Q4 `) j( G6 r) sAdipocytes
) S( | S" F1 ^6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation, S q% y" t" N2 H5 R
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
( X# K/ Q% f8 F4 v( I8. Unit 23.7 Neural Differentiation of Human ES Cells
- R) S, t" C% y# j) O* h' z" [. f26. Chapter 24 Lipids
, I) |$ C9 c) s2 U) b0 ?1. Introduction+ v/ p2 M$ g0 W0 Z. k8 m$ E
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking. T: u) j3 w" y
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
. s7 k' z l8 s3 m' k4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film* _' u+ a4 }0 J" |9 F
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
* h/ H0 n" [# g5 i27. Chapter 25 Nanotechnology
' k3 m# b1 g Y6 p1. Introduction$ @4 n9 s6 B2 J$ h( S/ S8 z( `
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes8 i! q! H, p3 H& ^! L+ m0 q- z1 T! {
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering; [- V o" o5 S2 n* a5 J' \4 a( q! L
28. Chapter 26 Viruses
# [- V3 W) `$ e) O1. Introduction
' F4 y, ^5 P$ a3 N5 u2 ], t( X& S u2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
: ~" D6 b/ p2 x: C0 T& }9 Q3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of- @6 U2 }5 I1 D4 C3 s
Cell Entry/ g, P* h: B$ U5 b# h
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection0 l+ i0 ^( \ X- |9 |
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
4 U; ~5 H o1 h% u h' L8 O6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
4 B# r0 l/ R& ]/ o6 b8 o. Z( F29. Chapter 26 Lipids( A+ x$ Q4 O B U+ [! E# Z9 E
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
8 W A6 H: r. VReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
( r& E" T: X# K0 ~* j& f# A! e30. Chapter 27 RNA-Based Methods in Cell Biology
- a) ?3 G4 |& ]4 v- B1. Introduction
) {: |0 E- x8 L8 I1 R/ c# ]8 Y: P2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs. ~& n+ {( r6 r5 e5 C, S6 Y/ e! }: L
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
+ _ G+ a1 Z. H: _/ GRescue in Mammalian Cells# R' d% X! H1 \& L* T, g
31. Appendix 1 Useful Information and Data# }, P6 r; I2 U2 e4 W! u1 i# T
1. 1A Useful Measurements and Data- L) }: ]- c' w3 ~ j; {- [: |- j6 M! J/ A
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research5 [9 M8 v0 T: I% Q& x% @
3. 1C Identification of Motifs in Protein Sequences
. D, Y( }- F D! x& D4. 1D Safe Use of Radioisotopes
' n. z S# U1 g( M5. 1E Absorption and Emission Maxima for Common Fluorophores
, e5 h4 t0 q S2 A6. 1F Importing Biological Materials
0 W5 [/ b3 h j1 U7. 1G Centrifuges and Rotors& o2 I2 ~1 \- _& Z# d* _7 P9 c
8. 1H Internet Basics for Biologists& n- }- C9 G1 \/ t
32. Appendix 2 Laboratory Stock Solutions and Equipment% h' a$ {* T1 h( t- \# z
1. 2A Common Stock Solutions, Buffers, and Media
) R# O$ O. t9 [) \1 R9 \2. 2B Medium Formulations- e1 H. ~1 c' [6 c" Z: [
3. 2C Standard Laboratory Equipment
8 [" A* T# I7 I& X33. Appendix 3 Commonly Used Techniques5 s; u4 q4 Z$ A7 u3 w, b
1. 3A Molecular Biology Techniques- e8 w- Z) b6 G% H% Y3 R/ F5 [
2. 3B Spectrophotometric Determination of Protein Concentration
: ^; U/ M& K# x( ~6 f" R% r3. 3C Dialysis and Concentration of Protein Solutions
N: l+ ^8 B, j% r4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
* H; V$ W9 ^& ~/ _6 }+ P5. 3E Silanizing Glassware
8 P" Y0 p% E( H$ J: k' ^2 E6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
4 E: j F W% F/ F! W7. 3G Micro RT-PCR+ r" X! ?( X6 U5 C# E
8. 3H The Colorimetric Detection and Quantitation of Total Protein
/ A S9 p" z4 D+ C& A. q34. Appendix Suppliers+ \% ^; l+ T) u. s
1. Selected Suppliers of Reagents and Equipment
9 V8 T7 q7 I& D$ K3 F) ]& e6 q. W: A8 R
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发表于 2011-3-8 15:53
