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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 * p! ?+ a) W$ i
5 l- l* h4 G* ]& G- C0 rCurrent Protocols in Cell Biology 2010年完整版 5483页* X; _7 G% ^$ z( _2 a+ p
! m' v7 A& Q" ]1 p! n: n1 m/ u3 D
Online ISBN: 97804711430316 [6 V& w0 G5 C
DOI: 10.1002/0471143030
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Table of Contents
; Z# z) E0 F ^5 k, u1. Preface5 ~! ]; ?- d2 y
2. Foreword
' @& _* ~2 U. a1 ~4 z3. Chapter 1 Cell Culture
0 W2 f; `4 V. c2 Y1 }3 J( C- T1. Introduction+ {! H+ l A- s) W1 K+ Y
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture4 M1 [$ D4 D5 N! n1 k- b
3. Unit 1.2 Media for Culture of Mammalian Cells
2 g& V: ~. g1 T$ t, O/ O& r4. Unit 1.3 Aseptic Technique for Cell Culture% s) C3 W' h, B- q- X1 B3 z8 H
5. Unit 1.4 Sterilization and Filtration
/ w- X8 `& ~+ \0 M* L, B6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
( J. Q$ F' ?" M1 r; K3 x& |% v, @* E7. Unit 1.6 Media and Culture of Yeast9 S7 v- B0 Z( A6 j
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
; Y9 U; F% p5 ]+ H2 k- o2 ~: q; i4. Chapter 2 Preparation and Isolation of Cells
( q/ O3 R! i9 F( t" l1 |1. Introduction
7 t7 T8 Z0 d" }2. Unit 2.1 Establishment of Fibroblast Cultures
6 p/ q6 R' e9 i5 }% h; @& c3. Unit 2.2 Preparation and Culture of Human Lymphocytes
' R R+ }/ B U0 [4 e6 Q4. Unit 2.3 Preparation of Endothelial Cells+ v' H8 E: B' E' S6 d
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
9 T9 V* a& _% W3 U: k6. Unit 2.5 Laser Capture Microdissection
, x8 J3 h& T- F7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
7 y- n" D3 k+ O8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells9 N/ n/ r2 C3 p+ H" `+ P
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
- o [: M. r( O2 U& u" M1. Introduction
$ o0 H, U# m% r# Q$ j9 B2. Introduction
; U0 g- v& k# {2 a6 C) j. t% G3. Unit 3.1 Overview of Cell Fractionation
; n% x6 R2 z, k+ | Z5 q- v7 m3 G4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains( _$ M# F- _0 x8 O2 q4 m
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
5 G" n$ G$ B1 t/ G- U6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
5 p" ]4 ~7 v9 K7 @9 x- ?; F7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient t, W; x8 b# S6 ]
Centrifugation
! _6 O' {" ~/ {5 `1 g. L8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient0 i* c, U# ~& d
Centrifugation R$ ]' V6 m# g6 r2 m0 ~5 W
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
4 M. x' S5 \# I, H3 z& U% L10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
' A$ U& ^. J, U5 ? u11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
9 g# _2 {8 z2 f! g+ f# b' QCentrifugation+ J" p, ~2 F: d& M* Y
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
$ d: b7 _4 A3 F$ _13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
' H9 y7 y8 j* r+ I5 o14. Unit 3.12 Isolation of Synaptic Vesicles1 J# `$ ^& R* y8 h, Z! {1 q
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation7 r' N( x$ \1 g& |" I6 M. i4 h
16. Unit 3.14 Isolation of Melanosomes
/ V! V8 i& A7 L1 W5 A17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
$ E1 a# r4 _$ Y/ `1 G0 P18. Unit 3.16 Isolation of Mast Cell Granules4 u7 E0 j% u. V! o; `( z e- N, x
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster' i0 ^2 z5 @0 o, F3 z) \# _$ S. Y+ ]
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas* Q( D. F L9 N ~8 z; N$ c
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons8 H+ J& \( E& i
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
2 l" e6 g$ `( u5 p( l! A23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes0 k2 ^4 H8 M2 l
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
: ?! j4 ~- V$ c; d* y3 C1 uFluids
5 B0 {1 J; h Q1 s- c25. Unit 3.23 Isolation of Intermediate Filaments
% V: M( d6 E+ I5 }! |: J- u H26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle# T7 }1 b) [) G6 N+ S, r
27. Unit 3.25 Isolation of Myelin
- Y5 B/ q+ x9 X3 ]& a% ^28. Unit 3.26 Isolation of Renal Brush Borders' h( a4 U5 o( y& Z
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
/ [' T# U' f( w( @ y* _1 RFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts0 i6 \+ H! H" ~
30. Unit 3.28 Isolation of Amyloplasts. j; R t' q& r1 \) v
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
) O+ G/ y8 a6 X+ @0 t5 _2 i4 ^" M: |32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
* N/ N1 m1 w8 D5 L: p$ h; WCentrifugation
s! {: T# G7 c' l7 {# ^! B33. Unit 3.31 Isolation of Neuromelanin Granules
+ @6 k1 x H: p9 }( O! R4 w34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and R8 l8 U1 |: n, E8 ]! _ H; ^1 f9 n
Density Gradient Centrifugation
+ t8 v& a3 B7 T- `" ]- q; J35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
3 n& B0 W# W5 ^3 r36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
7 i7 }7 V M& M37. Unit 3.35 Isolation of Platelet Granules- b% @1 D: a" W. @/ m5 ^
38. Unit 3.36 Isolation of Nucleoli* f/ K$ g) V6 S, l1 P8 E
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
: I. W9 d2 [8 y r6 w; p, W& c3 l3 F, C40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
7 e7 N$ E4 E, _/ d5 Q2 g41. Unit 3.39 Isolation of Chromaffin Granules
' ~- v& h" q6 W5 v* n) q42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
5 z7 j6 [7 N! q" M3 t0 w6. Chapter 4 Microscopy
0 j5 U) g0 ?" O% U- K# y/ j, J8 Q1. Introduction' T! @( m' G$ e5 J+ J
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope( M. U# e! z% D8 ^! C& z% M
3. Unit 4.2 Fluorescence Microscopy
! H7 }* w) p4 Z) \8 h8 m9 ~. |5 e. R4. Unit 4.3 Immunofluorescence Staining
9 Y" K E' I, K ^5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
) O0 `3 I1 _, A* t4 G* {6. Unit 4.5 Basic Confocal Microscopy* B6 u' E* i2 _
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
) a, I# l9 ?& `$ Z: T3 O8. Unit 4.7 Cryo-Immunogold Electron Microscopy
) h8 K0 R% y3 i9. Unit 4.8 Correlative Video Light/Electron Microscopy' {7 i* N, _) p. u
10. Unit 4.9 Polarization Microscopy7 K. v, K; ^% h
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
1 R M# }8 e. ~$ ]( D8 `12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues+ `4 p" |5 X0 ^5 l
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
; P7 {7 g2 ^. D2 E# X# j8 hEvents1 \ G; q9 P4 ~0 y; O+ Q5 f& r. t
14. Unit 4.13 Fluorescent Labeling of Yeast
; x' o- x9 [& C: l" u/ ~15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
; _3 ~6 b4 W4 y& [& Y16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
# z+ p1 W. S1 W% ~" s" U17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules ^9 X `; D1 Y& c
in Endocytosis: b! d! {( b& @$ E6 g
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
% X+ P: `% q' _2 k19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
8 V% G' y/ B. T1 S; [0 f20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images8 c4 q+ U: W9 H- G) B
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions+ I% S! s) P& d. E' `" T
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes$ x5 i$ o( @% k. p* Q1 w% W
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics3 }+ t7 z. r1 b) Z9 {1 T
24. Unit 4.23 Interference Reflection Microscopy' J1 R! \" D( M5 m1 W! j! i0 L
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
v* c9 L" p1 \' _/ d26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
; g2 N5 b" H/ c2 M Y$ }" a27. Unit 4A Organelle Atlas: Appendix to Chapter 4# q; J! V/ Y. C
7. Chapter 5 Characterization of Cellular Proteins
+ t6 p; A9 e) T& u* X' j4 n1. Introduction
) O+ e8 S/ d* e$ Z; Q# O) T2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
7 \6 v7 M8 x! N' }) X8 T( N3. Unit 5.2 Determining the Topology of an Integral Membrane Protein N' V% n5 u; N1 }7 ]1 ]0 v# M
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
0 {* G4 [0 X7 g- U+ F0 h% h5. Unit 5.4 Analysis of the Association of Proteins with Membranes( S" A" Z$ }* c$ O ^
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
/ D& N; G I) n8 X7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
# i. w; A! X. oSpectrometry+ l. M& g( E4 W' Z$ z2 t
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening+ x6 {- q2 ?# U: Q( j$ }9 c+ z
Applications9 l; X; ^) f" u7 F% M
8. Chapter 6 Electrophoresis and Immunoblotting6 ?# J7 g9 H, S% w% a5 y( ~
1. Introduction
! {) s& S$ ? B: J8 A w7 f1 L2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins7 U6 |3 o; c3 A0 ?- ^9 [ S
3. Unit 6.2 Immunoblotting and Immunodetection
5 O; m, R( N S; s; u3 }4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
* N* H, w. s0 _( j' h: G7 `! @5. Unit 6.4 Two-Dimensional Gel Electrophoresis5 x' C. W- {. }, H
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
: b/ K O8 }- x3 r! m; e8 a. \7. Unit 6.6 Staining Proteins in Gels
9 D3 p% j2 s5 W0 ~5 ^% h* `8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
1 J" k6 u; k6 R- ]5 n9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots& E) Q5 Z4 U; o! b7 M
10. Unit 6.9 Digital Electrophoresis Analysis
' ? ^( M* h1 C" F11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis8 a9 W/ F5 o- @; S7 U( v$ g6 t- M
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of8 N$ q! n" j" c% {
Single Cell Gel Electrophoresis (Comet Assay)0 t+ D/ K) `4 a7 B. I# c
9. Chapter 7 Protein Labeling and Immunoprecipitation3 d% S( h, K$ K
1. Introduction4 Q2 B: L" ^) U3 S Q% j
2. Unit 7.1 Metabolic Labeling with Amino Acids3 L% @' V* e6 ?0 P# A* T
3. Unit 7.2 Immunoprecipitation3 k( ?- ~: [, G q, n+ Q
4. Unit 7.3 Metabolic Labeling with Sulfate
: v& P, G2 q( M* {, B; L; L5. Unit 7.4 Metabolic Labeling with Fatty Acids% H1 c( Z! s/ F4 J# D' r. B' b
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
) p6 [% |$ N/ J$ [ P1 X! s/ V7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins( k ^. u: @+ U8 \
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
- r' Q9 u! H( L$ o2 t. r. N0 z# J9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars+ I9 a! N/ d6 `0 r4 K
10. Unit 7.9 Analysis of Oxidative Modification of Proteins' @- C/ P" s$ ?
11. Unit 7.10 Radioiodination of Cellular Proteins5 Y, F) {3 F) I0 j. I- j
10. Chapter 8 Cell Cycle Analysis
3 j7 f, o& h3 r1. Introduction
( C6 F2 ^. Y4 k* e% E. k# J5 m2. Unit 8.1 Overview of the Cell Cycle* o% Z$ {, V$ `6 s% M
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
# f7 ?& Y; C/ {9 R! \4 @5 C# x4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
/ Q9 T; i, i1 b0 z! w+ u7 n h2 T5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
8 Z( ^. l0 T% ?' L3 _6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells3 y* ?' Z: Y4 Q, b/ Z
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
7 J+ F1 C( f% J8 h11. Chapter 9 Cell Adhesion8 a6 A* N- r* \5 M9 L- B5 R/ P
1. Introduction% R7 |* y% D |$ n9 [! z0 Z
2. Unit 9.1 Cell-Substrate Adhesion Assays
' Q! w( N& V& x7 l9 m3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force) T+ F- c7 N- J+ B8 R$ V( a$ u
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion( W" p4 I7 T9 s6 h6 A+ G" V+ @
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion5 J% b, b8 R) d3 w
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules; ?4 {2 Y0 I* _, m: _
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions7 Z6 I8 m0 C. S9 q* l
12. Chapter 10 Extracellular Matrix' x# s5 u' m9 Q
1. Introduction* _& M6 ~ n: z& V: M: ]( l+ Y
2. Unit 10.1 Overview of Extracellular Matrix! H# h5 l& M9 i" a
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
/ V. o' S* r1 a7 F6 |! B+ [4. Unit 10.3 Preparation of Gelled Substrates1 R1 u% y4 u7 C+ L3 k
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9$ C2 y4 \! q6 t- F r7 ?% K4 h
Endodermal Cells
+ v* F& a% J7 L% }' ^6 g7 N6. Unit 10.5 Purification of Fibronectin
% q; x3 R1 {& \* m- |4 |8 w q7. Unit 10.6 Purification of Vitronectin
t/ \, T2 Z- A8. Unit 10.7 Proteoglycan Isolation and Analysis- E( X9 T1 P2 @# D, z. D
9. Unit 10.8 Matrix Metalloproteinases
) k' c) k# q$ T5 b! t10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts6 Q& e# e& `" \- N6 t
11. Unit 10.10 Purification and Analysis of Thrombospondin-1+ r# s$ [& I# t) ^
12. Unit 10.11 Purification of SPARC/Osteonectin* G( J- a7 g1 J/ ^' n+ u! {
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly# e0 Z% q T3 W1 T
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
! g" Z/ R( O" y9 F" t4 ]15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor8 o, A6 C, m( Z6 U* O5 b
Xenografts
) J6 A. y4 `7 P* c" i16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning& g t, e4 n, E: V" q9 W4 @
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
. F! E1 a1 G$ X" ?* l6 u* `18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D* @% J2 X) ~0 f1 {0 V$ V# \0 E
Cell Migration; R& ^: w$ \/ D) y, @3 r
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
7 f9 y* l t: }( s: S2 u13. Chapter 11 In Vitro Reconstitution5 t( d0 I/ l' e3 `3 U$ W
1. Introduction! D0 v4 m0 B& k, a& A1 Q. p+ J
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems# u- e4 \7 q" ^0 P* z3 h) G
3. Unit 11.2 In Vitro Translation5 {- V/ d. F0 `) D- @* W
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
9 L, o9 ]" y& `: M" n5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
/ D; p6 u7 j0 w) d$ u6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication* D& U" s0 U$ z/ V
7. Unit 11.6 In Vitro Transcription0 v' Z! T8 j+ c( c
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
" G$ o3 n; C8 a: {5 c6 I# M& {9 w9. Unit 11.8 In Vitro Translation Using HeLa Extract, O; g5 C- M; i4 ]; Z W5 v
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
0 I5 t2 h: y. G! T: Q9 ^; u' }11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 ?' C; d9 l4 X8 v: b
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts k2 N6 @! i" L2 F
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
8 K/ c% o9 B) ` P- L- t. w14. Unit 11.13 Mitotic Spindle Assembly In Vitro
* e1 n6 Q- {2 j$ n/ V15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
. E$ L1 b" i# h8 j, X# P16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
; k9 a$ S- P, h9 l3 J9 e* b+ s17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import( M& H; F2 p9 Q8 j$ `
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts1 Q; g, B/ r, n1 n& D8 ]
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
5 l; f" d& u! t: ]20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
8 u2 ~$ s3 x" ?0 i; p- L14. Chapter 12 Cell Motility
& f! x7 P ]% M- x1. Introduction& b9 P; f& F* t% S% E, m2 c; l1 J
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
* {/ Y$ S/ H3 F# R9 Z3. Unit 12.2 Invasion Assays
7 Y4 L' Z! v* p" I- f4. Unit 12.3 Cell Traction6 \8 }! _2 e( m+ o, z
5. Unit 12.4 Cell Wound Assays+ N: ]9 K3 m) R' H i
6. Unit 12.5 Dictyostelium Cell Dynamics6 f' y* v3 k/ g& A( B# [
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils2 W, S# W9 S: Q ?) i/ y3 C
8. Unit 12.7 Actin-Based Motility Assay
1 J8 E0 O2 i9 f' X! U1 H9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP/ d8 b- y" c; S7 r
15. Chapter 13 Organelle Motility* ^ m3 K6 Q5 v7 K: E0 T1 j
1. Introduction) k7 G5 P- u( \( [
2. Unit 13.1 Microtubule/Organelle Motility Assays
/ k8 B$ u2 t9 e* J4 d; \! A3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin# ^* n( j; n; J' [/ Y0 Y
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
* ~1 ^/ d, M: N q, h7 a5. Unit 13.4 Movement of Nuclei, ~9 M& y8 M% I
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching7 \1 C) D& G0 {9 w; d3 Q. e2 N
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly* I/ u( q- V. ~' `( L$ `
16. Chapter 14 Signal Transduction: Protein Phosphorylation
3 G2 H3 [& C+ {( }2 `1 G1 p3 s1. Introduction
, E9 _" H. d9 u) {! N" g2. Unit 14.1 Overview of Protein Phosphorylation8 s( w7 x9 L; E7 g) ]3 X9 z0 c! E
3. Unit 14.2 Immunological Detection of Phosphorylation- I& c+ H B6 ~$ t
4. Unit 14.3 The Detection of MAPK Signaling
" f' r- Q& C s) w8 p- ? G5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation8 w& j: M. x8 q( ^/ n3 H
6. Unit 14.5 Phosphoamino Acid Analysis
$ I: @9 ]- Z+ Q$ M, r, W% g! M z9 Q7. Unit 14.6 Determination of Akt/PKB Signaling$ Y' h) |# G6 @1 t- a
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
$ p4 E/ O2 U( S7 s& B9. Unit 14.8 Rho GTPase Activation Assays
2 T# Y4 W& x* v/ V3 O1 [0 ?, e10. Unit 14.9 In Vitro GEF and GAP Assays, y$ V9 S6 f3 D0 t3 l, |3 z
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
" x& ~2 t8 f( |2 n' E8 ?Energy Transfer (FRET)5 N- O1 C% m* t) t1 F6 W
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
+ }) @$ @) ~- G8 j3 E( J/ `13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells8 P: k u, y1 n- T" w
17. Chapter 15 Protein Trafficking
; Z. a+ m4 G# U) w# L1. Introduction/ E. e; A' J" ^* s/ y
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways7 k1 A) r( n( X- T3 C& S5 M
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
% [, R$ g3 ?6 q, S* q4. Unit 15.3 Endocytosis: Biochemical Analyses8 G) i% k8 R+ B4 l- ?2 q/ {- ]
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane7 p5 v7 r ]- V+ z
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells# n4 R! {7 @2 G0 U
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
) i6 W& l# Z$ L1 e; s& X% e8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
; S, i. p6 ?8 Y c `9. Unit 15.8 Analysis of Protein Transport to Lysosomes
' f0 b7 I6 f) p10. Unit 15.9 Studies of the Ubiquitin Proteasome System
& P+ L F* Q6 v- o, _% A11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network; Q7 Y; m$ i7 n2 V6 Q, K; Q1 `$ k
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
* \$ I; E( L2 D p13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells- ]) y( L# Y) I( N: r2 Y/ W
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
( {& g, \1 R$ |; ?$ B15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
* I: V+ ^( V& r3 X) }5 B( a. lEGF Receptor
! M( m7 d8 f/ m' J$ O16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
* w/ x, @7 e8 X p" E18. Chapter 16 Antibodies as Cell Biological Tools* X1 p$ s3 Q$ p; G0 E. f7 o
1. Introduction
4 h$ X0 M) O, b- u2 \2. Unit 16.1 Production of Monoclonal Antibodies% f1 ~8 H: |0 A. X
3. Unit 16.2 Production of Polyclonal Antisera
) R3 j8 `4 p0 h& _' k) t4. Unit 16.3 Purification of Immunoglobulin G% g5 e+ k- Z" u
5. Unit 16.4 Fragmentation of Immunoglobulin G
3 ~( V' I6 q. L! [6. Unit 16.5 Antibody Conjugates for Cell Biology; [0 Z! _8 ? I
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
5 g5 B) T3 l) z$ [' Y; q- i( H! ~19. Chapter 17 Macromolecular Interactions in Cells+ n# i0 r1 c, v* t
1. Introduction% `! }+ I- b3 i- A- X# A. r4 L1 K
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)3 n N( f- }5 b& n" [
Microscopy
0 @& s' e. W8 p' L. `3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis7 w, m5 [" Q: R! _7 s* b
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins7 x# U$ q" c1 V$ m1 y( C
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries& y$ J3 D9 g1 r" b- U9 i, C, z4 j# ^
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
- M# a; E+ _- `& c3 V7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
; T c3 q: ~" f8 H8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific! U4 o; F+ C f C/ d* P
Genomic Sequences In Vivo2 i g, O2 F! M/ l
9. Unit 17.8 Isothermal Titration Calorimetry
7 m; {3 Z. `/ ~8 ]+ V$ R4 _1 d4 `9 r10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
9 b" T5 e' a$ I! J7 y) y) s. y11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking8 ^* }% V* e n. b+ r G8 s
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
$ c( m2 X7 C' Y( B0 h) SInteractions (RFAP) in Live Bacterial Cells
: f6 _' L* K e$ f$ U9 } u8 d; C20. Chapter 18 Cellular Aging and Death
. s) I( x. C: z/ N9 m, a1. Introduction
Y) T# B: e3 }* q2. Unit 18.1 Current Concepts in Cell Death. o2 d7 p$ j+ ^+ G; A5 @2 g
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis+ c" z' B. R# W3 X2 d
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria4 S8 j2 J& U5 A" A! K ]
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
; c2 c( h8 I8 m Q- P$ R6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
! L' O D8 p- P9 t/ J7. Unit 18.6 Analysis of Telomeres and Telomerase
) a' V0 n; G% p8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of0 l& |, [2 r2 p/ P' n5 ?1 D: i
Poly(ADP-Ribose) Polymerase) q; t3 j, i" s5 v# i9 @2 D3 w
9. Unit 18.8 Flow Cytometry of Apoptosis
5 [& N) V8 `# Y& m5 \10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
, W+ n+ F. Q5 B) mAssay% I1 d' c9 d" Y/ P3 ~/ q
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis. Q. t, \" V& f: o
21. Chapter 19 Whole Organism and Tissue Analysis
: b; Z, v2 V+ T' w+ ^1. Introduction1 x$ Z9 b3 x/ s- d* Y
2. Unit 19.1 Overview of Metastasis Assays
9 l/ O5 d9 h7 y, U3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
" v9 s8 N6 v7 B+ u! p( k4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene! p: K4 C. F7 ^1 W' n( \5 m+ h
Expression After High-Fidelity T7-Based RNA Amplification
' e+ \! Z* u6 U( T7 r5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA I% V" I; E0 a. w( Y+ ~% y$ B
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
; s" Y$ V7 e; o+ B4 z! a7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
( L. L1 ^0 Y6 V7 m! S* E5 `( Q8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
" x* h- V* S$ x1 `; v9. Unit 19.8 Embryonic Organ Culture
- L, r, @6 |- U& B10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin2 Y1 K: I3 S" ~: ~
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
. }; d6 N$ Q& R; B2 ~ U% P12. Unit 19.11 Generation of Transgenic Mice( V1 w4 Y: r" G2 U
13. Unit 19.12 Overview: Generation of Gene Knockout Mice
! t/ m- X% W5 M) ?5 h14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
G# t& j; D. `9 r15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
* ?1 [" ~- x; X C- D" C22. Chapter 20 Expression and Introduction of Macromolecules into Cells
+ i( u$ ]7 y" i4 \8 w- m1. Introduction1 C4 K+ A6 V. e. y9 _7 ]! E2 I
2. Unit 20.1 Direct Introduction of Molecules into Cells
' [' e% \2 ^$ D5 T, N3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
; u" S5 J) C- Z$ }! }8 Z4. Unit 20.3 Calcium Phosphate Transfection
W; x( h. S' w2 \5. Unit 20.4 Transfection Using DEAE-Dextran
( g# o5 a: G( j# z, G0 f6. Unit 20.5 Transfection by Electroporation9 e' V6 B6 S1 ]- F3 J6 @" D
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents h. ^1 z- s1 b# G
8. Unit 20.7 Optimization of Transfection
" S: F% Y: G( G) E8 [9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
5 h0 ^ D7 f& v' _0 ^7 y23. Chapter 21 Fluorescent Protein Technology
5 i+ @0 \6 T. f1 T1. Introduction3 u. s* G4 V1 a* D& E0 F
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
) c6 c9 w$ U( \$ a, s# L4 _3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
, ~" M# z& S: c4 Y4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence) ~. C+ Y1 M5 h! R
Complementation (BiFC) Analysis: L) p1 z, z- u( {: B
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
( N, h5 ~8 b2 L# u" F) h6. Unit 21.5 The Fluorescent Protein Color Palette% i6 m- o" t4 y q1 @4 r# s/ U; `
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins" n9 s, |' q: h6 ?) j/ B0 T/ e% W
24. Chapter 22 Cell Biology of Chromosomes and Nuclei& `1 [% C9 i, q, ^
1. Introduction6 d$ |% s( X, Y& R+ u
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
( I0 C: _( B+ _3 u. q, Y3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples$ w1 z# S; D/ v7 x3 B7 [# v3 k& U
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis5 P* P" \- n6 A0 ]/ S
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)) z& x) x2 n) W5 N7 v2 ?7 p( ?
6. Unit 22.5 Multi-Color FISH Techniques0 I& }2 e3 j8 |, f. Z
7. Unit 22.6 Comparative Genomic Hybridization
/ Q! u6 s1 P8 J; W8. Unit 22.7 Sister Chromatid Exchange& ? A; h7 O0 r+ P
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
9 n& g$ Z* k/ ~0 i3 D9 V( o' Q10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes* w% |, M( _4 U: p- R* _7 X/ G
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
9 W) O+ G; r& e* m6 n9 k7 }12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
2 G, S3 [6 a; w0 U% f/ E# m. a13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
: e7 N$ K2 r, _, g14. Unit 22.13 Monitoring mRNA Export
. r! D4 d0 g; l! C. a2 n M! Z15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
, D5 R5 z; @. o2 JField Gel Electrophoresis% N1 U3 U, {* R: M( C& f' `$ b1 v5 V
25. Chapter 23 Stem Cells5 @! t$ x5 A( d& b/ U" s C
1. Introduction
/ o8 p& l& T. X* @+ z/ R$ b' G# H2. Unit 23.1 Stem Cells: An Overview
+ e7 F; g8 ]5 J* n- f9 ~# I# M3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture7 C; {/ S$ R# P8 q' W. X+ Z! ^
and Differentiation as Embryoid Bodies
4 z: n3 o$ f, e7 l2 p2 x/ P2 C8 S2 T8 K6 `4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
, U0 [5 U) g" ^6 @5 CVessels) b- W+ [) {( }/ B% g$ O
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into/ y, G& S% Q6 N# c9 x, e
Adipocytes
( L" A# U5 d! e) _3 i# W' n( {- U' e6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation& ?( D+ w, ?0 ^/ g
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells* w9 z/ c$ W* W x L! F8 W
8. Unit 23.7 Neural Differentiation of Human ES Cells
) ^" B0 S1 M+ P& P$ S9 B' z26. Chapter 24 Lipids4 v: x# E, _2 ]; w
1. Introduction
?4 N% v5 e: e( ^ y, G2 d2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
* c* j& @, O. E3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
! _: k. p* L' s& L r. Q/ v x5 h4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
0 y2 n/ \0 r- r! O5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
. i; P: Q% B6 Z6 T2 i* S: g27. Chapter 25 Nanotechnology$ T. K2 L1 o9 a% A' O
1. Introduction
# w5 X5 W1 Z7 b9 w2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes. ~+ n- q% U! y, |
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering2 M/ Y; b8 A( q) L* \
28. Chapter 26 Viruses: S0 o3 ~% p% N6 \2 j8 d
1. Introduction& Y& R3 y' ^" G
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors. M- O. u1 [& ~& g% H
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of2 a0 ~: E1 S( q G7 v4 v
Cell Entry
6 Z0 B3 \, e' I5 `4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
1 ]9 p2 |1 X& B, b* L/ ~5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses; P1 o+ ?2 b7 N: h2 Q% m! k/ I
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission* T' M) ~4 H+ e$ n% w! [
29. Chapter 26 Lipids0 K3 Z, @4 K0 Z
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by- |# F2 ~$ [, U
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
3 r# u% {+ n0 Y+ T7 f30. Chapter 27 RNA-Based Methods in Cell Biology
$ w, ]3 x2 t' b, v2 y! o1. Introduction% ^" H( Y+ U5 v3 V
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
$ P, L' J6 V& @& d- T3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible1 B8 |& D. E+ r$ c7 }
Rescue in Mammalian Cells9 _1 i! y4 P7 L, F) c- R
31. Appendix 1 Useful Information and Data
* m1 ?) j9 p R" ^7 M& i1. 1A Useful Measurements and Data
! B- Z O6 r2 o# X b& Z' P2. 1B Compendium of Drugs Commonly Used in Cell Biology Research6 `8 _) S- p) p* W# q+ c8 F
3. 1C Identification of Motifs in Protein Sequences- j1 C4 q( X7 E/ E2 d5 K
4. 1D Safe Use of Radioisotopes! F2 u& Z3 S8 n
5. 1E Absorption and Emission Maxima for Common Fluorophores- [+ c9 R( {( M* T
6. 1F Importing Biological Materials; s6 L" {7 y7 q+ V
7. 1G Centrifuges and Rotors
" f( t* e7 R! i* i' T8. 1H Internet Basics for Biologists
) ^, e- T3 F7 O/ n& @$ M32. Appendix 2 Laboratory Stock Solutions and Equipment1 x0 ~ r* @0 V0 _+ r; [ @
1. 2A Common Stock Solutions, Buffers, and Media
: t' F9 H( d0 W* v6 _, g2. 2B Medium Formulations
' Y/ ^4 D3 ], f% a3. 2C Standard Laboratory Equipment7 W+ r; X% G2 m, x( ?
33. Appendix 3 Commonly Used Techniques
; D- o+ Q# e" V5 T4 r# L1. 3A Molecular Biology Techniques
: D+ `7 r0 H8 r( f8 t5 A* z2. 3B Spectrophotometric Determination of Protein Concentration" k$ n5 e2 z5 B* h" u
3. 3C Dialysis and Concentration of Protein Solutions( }1 B `' L% W
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy$ A: h, \3 C4 T/ h; L
5. 3E Silanizing Glassware0 K' Z. x, x0 {6 ?& z4 J
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
5 Y+ ?4 d' [8 ^; l% e# i7. 3G Micro RT-PCR5 d5 D, s& j% N
8. 3H The Colorimetric Detection and Quantitation of Total Protein
: N5 j$ b1 @3 f34. Appendix Suppliers" x' W0 k0 i0 z: y5 ?) B+ O: C
1. Selected Suppliers of Reagents and Equipment
3 @/ b5 U5 g. ?; ~6 C; z' \0 D! C; F D/ J3 @
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