PDF电子书：Current Protocols in Cell Biology 2010版

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Current Protocols in Cell Biology 2010年完整版 5483页
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Online ISBN: 9780471143031* d( W2 x9 H) `2 S% j* |; \
DOI: 10.1002/0471143030. k- [: h4 D: A! g

% H  _) e7 _. S0 [! j3 TTable of Contents2 M+ ]+ n0 i2 _- U
1. Preface
; s) F- e9 t1 ?- ?8 ~2. Foreword& j. Q* v& f& u+ b" f
3. Chapter 1 Cell Culture) Y- x6 A8 d3 [/ J8 r% f
1. Introduction
- t6 O; Q6 i9 g2 Q2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
! D) T" B5 J4 Z& ^1 e6 N- {- S* F3. Unit 1.2 Media for Culture of Mammalian Cells; z0 `- E2 V4 V0 P- l# F: }" C
4. Unit 1.3 Aseptic Technique for Cell Culture
( f  N9 J/ z1 b9 {, Z; R3 w5. Unit 1.4 Sterilization and Filtration
2 Z; P) I3 S% ?, |% I0 z6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
$ F- t0 _. @$ J% F7 {0 g! X7. Unit 1.6 Media and Culture of Yeast' m! W, t8 ^! i; H& x5 n0 g2 v
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
% y9 y! R6 N# h; _4. Chapter 2 Preparation and Isolation of Cells
, o" v4 O$ S# V; W  C9 T  c) L& @: k1. Introduction& f1 H* D' b% u3 c' R
2. Unit 2.1 Establishment of Fibroblast Cultures& L: A$ {/ {( _/ P6 w
3. Unit 2.2 Preparation and Culture of Human Lymphocytes1 @3 p0 H- l$ t' L0 ^# @
4. Unit 2.3 Preparation of Endothelial Cells
' q- K  }& ~$ G/ o/ s  K5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation& Y2 Y1 c8 J$ W2 E" Q; F  {
6. Unit 2.5 Laser Capture Microdissection6 u; B' `4 [, \4 T
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
$ j) }2 f2 f# v3 m4 H' s# |; c8 l; U8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
3 D/ H6 g6 F* f' U1 B5. Chapter 3 Subcellular Fractionation and Isolation of Organelles* {9 O4 b# Y8 Q$ ^3 V1 n! b# _
1. Introduction" k6 ^. J4 y0 K$ U* L  V" G0 q! F
2. Introduction: C. i2 Y/ q8 E3 x6 g  e7 ]
3. Unit 3.1 Overview of Cell Fractionation1 K, g6 `. k- e' R
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains! c% z6 F. m' \  w; O; [; S: v
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
$ [/ E0 n: @0 R3 B6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
* b% k; e, L; {- s9 z7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient3 l1 ~5 }' G/ a2 Y0 `9 ~
Centrifugation( }2 D( k& Z% V+ d7 y
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
* f( W* {9 X' t6 Y% M9 kCentrifugation. f+ d1 R: y8 h$ {0 A9 ?
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
# Z+ w+ ~+ G3 I! L10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae3 @% C# ^  @9 ^$ H  A. {
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
/ F0 i, ]' p& d) C+ vCentrifugation
5 }4 S/ _. i. T/ `6 ]12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
2 |4 j8 D: n$ b2 V7 O4 p' {13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
; b2 v3 B2 L) R9 S! L14. Unit 3.12 Isolation of Synaptic Vesicles
9 o$ m; Y+ {, ?: i7 I6 c" T% h15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
) d4 V6 q5 z$ t. m6 |16. Unit 3.14 Isolation of Melanosomes0 w" l/ X- J7 C* s' q" q- A
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation+ I( c& ~) j$ s9 L0 W( s8 B% t6 K9 |
18. Unit 3.16 Isolation of Mast Cell Granules; `9 |+ u- ~: A. o3 N5 R5 U
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster* C8 P. Q' s. u2 M5 _# _+ s' V
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
" V& S0 B1 h9 ]  j6 A) C. ^21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons; [9 |  k$ {  X4 \7 P7 h5 }
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles: e4 A% r/ x! ~* d
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
7 ?2 j. ~  Z  p5 U% h- c8 i* b24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological3 F' Q( n% L2 N7 ^) A. v$ e
Fluids4 I7 L  r' @  c8 J, `
25. Unit 3.23 Isolation of Intermediate Filaments2 D1 W( C0 E2 G2 }: O8 a8 o
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
( H" ]$ C. J& g( U8 w27. Unit 3.25 Isolation of Myelin
: }2 z, p) G2 u28. Unit 3.26 Isolation of Renal Brush Borders( l) j& m& \4 }
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane9 u' \: `) L3 G% k, u, h6 ?
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts* Q: e- ?7 ?% F% M3 }! U5 T; _$ g
30. Unit 3.28 Isolation of Amyloplasts9 {; ?: X1 e9 n8 Y/ m: E4 j) L* B3 O* W
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
, N/ A) [0 B( `8 b32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
% |6 }9 T4 D& L- vCentrifugation! a6 I! n! t9 s& u8 S
33. Unit 3.31 Isolation of Neuromelanin Granules, [3 v9 V- B8 N7 I; q4 C2 v7 x$ ^
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
, b8 @- @/ W+ ODensity Gradient Centrifugation
* a. }+ Z1 |) ~4 p& k35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments( v1 h# k" G$ O" S) ~/ S
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
, l1 d7 Z$ N: W: _: D37. Unit 3.35 Isolation of Platelet Granules5 h" F' j- ]: q9 [
38. Unit 3.36 Isolation of Nucleoli: A, W- e5 {" `0 f. n! O
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins3 I3 `# B: y2 A$ w
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates$ a3 F. }& ^( W0 R4 }
41. Unit 3.39 Isolation of Chromaffin Granules1 O* _; C: Y, g) M0 C
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines- ?9 `/ V1 l# q: Q
6. Chapter 4 Microscopy
9 J7 ]; W4 {$ x8 k3 M' V1. Introduction# \2 N* x/ y9 s9 ~1 I4 H' h7 e3 |
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
# W% e/ @& g9 k/ v1 {4 E& c) S3. Unit 4.2 Fluorescence Microscopy; k. }6 P& Q* x: V
4. Unit 4.3 Immunofluorescence Staining! Y( g7 e) O( d1 \& {2 s
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria' [" q2 u6 o. M* @" Q
6. Unit 4.5 Basic Confocal Microscopy
5 e% ?! g, n& j7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
5 O, }' ]! n, B7 a% K. ^0 p8. Unit 4.7 Cryo-Immunogold Electron Microscopy
$ z: ?2 O8 p8 z6 A9. Unit 4.8 Correlative Video Light/Electron Microscopy
$ x+ e' |+ s* X/ _7 _10. Unit 4.9 Polarization Microscopy
( `( r- D* D: q8 l+ \! @11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells" P: W( z/ o# |5 p
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues4 I: ]3 K1 G. s4 P9 I% `; a
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
1 R' W$ W; ~6 ~, rEvents
5 E& e5 A) w4 o8 o# }1 N14. Unit 4.13 Fluorescent Labeling of Yeast
9 E7 H4 P+ J( j: M, `15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
$ R0 \* c/ k- a: ^: C, [16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
! S# [( j9 {9 P9 j4 ~- V17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules; i! W* P" I4 A
in Endocytosis
3 s3 y$ T# B* {2 [- [- [7 |18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology& r* U$ ~, S) W( z+ ]7 j
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
8 b) T% b+ \! y2 @: b20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images4 P9 d% a5 r+ Q" }$ q  B
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
: Y6 w- [' i5 t$ V4 m3 c( y22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
! A* C/ O& n8 P23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics. K5 `# g( y8 s  H
24. Unit 4.23 Interference Reflection Microscopy
# ~: ]) V- W, M; x8 I9 b; }25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
0 i9 m! f4 ?: z) V1 ?& L) L26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches4 t) G) O) c) T7 n3 s* d! r4 }
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
5 W5 O, l% P" Q+ v7. Chapter 5 Characterization of Cellular Proteins
3 ^2 X: D/ y- z) H- o6 x1. Introduction' }6 j5 f3 k& x2 S% {6 f5 I
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells8 ^1 L/ _7 K5 Z0 ~1 y) }+ B) I
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
5 b9 ~. ]+ [3 f1 g: ^! v4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients  P$ g. P& Y! J1 N
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
6 |6 e! g0 i% N8 H6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration); x$ i  D; g" k
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
& Q/ g: @% K; J8 X9 dSpectrometry% n+ s( ?+ B) w, W# b
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
- V; s8 A, f# V% \; x3 q, t* F7 VApplications
  }" C/ ^9 \. {; W- d8. Chapter 6 Electrophoresis and Immunoblotting
7 |+ \: A5 s% u2 l8 Y& d1. Introduction) j: \5 f- v* j
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins% i9 I5 V5 P1 p. C& ^) o% [6 U
3. Unit 6.2 Immunoblotting and Immunodetection( m- l5 K3 O% K' x7 l0 F
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots0 o$ o& x% ~( T, T6 t0 U8 }( V
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
* F- Q/ a" Y9 E" {6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
+ u: M  H% F# z3 D/ f# A% n7. Unit 6.6 Staining Proteins in Gels% [' H# M% _! G7 `: O
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
7 j5 D. B+ C0 ?3 H, `# Q9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots) q* x6 Q2 ?. y& M: C
10. Unit 6.9 Digital Electrophoresis Analysis4 ^& g) c0 O- R* C! }% I
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
: b) Z6 _9 f1 p- x% ^12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
( k7 `4 t& D0 t" uSingle Cell Gel Electrophoresis (Comet Assay)
) {1 \' `, b# z! C9. Chapter 7 Protein Labeling and Immunoprecipitation. u: g$ |: |/ d0 \/ o
1. Introduction9 p: D1 a8 i' f, s+ w
2. Unit 7.1 Metabolic Labeling with Amino Acids  A0 O5 q# G& L  B( c
3. Unit 7.2 Immunoprecipitation
6 M' V9 Q. Y& N6 X( v; \- J4. Unit 7.3 Metabolic Labeling with Sulfate
5 d6 t' E0 y' T' Y5. Unit 7.4 Metabolic Labeling with Fatty Acids8 J- f# f5 r  N5 v; v
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups8 s. D! {* r/ v( x; v1 p2 H2 t- d3 ~
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
2 u6 Z4 u1 @9 l$ Y8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
- [' r. R. s" a# @; h9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
+ e7 _& f, ~0 g+ t1 n, J10. Unit 7.9 Analysis of Oxidative Modification of Proteins
7 Z: \+ F0 E7 [+ o: s4 t, ?11. Unit 7.10 Radioiodination of Cellular Proteins
/ |6 [# Y. t; v: L9 \) d- a10. Chapter 8 Cell Cycle Analysis
2 @, H! F; }. C$ m$ d0 Y1. Introduction
9 H9 Q! _$ I8 v" i2 C& ]; D% m$ h2. Unit 8.1 Overview of the Cell Cycle" b; o5 J9 q1 i$ W, z( v! v
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle  |9 E% i) f" |9 \+ z: j! F
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle2 G# K3 A) @" {+ W
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
3 Q, r: ^' r: f9 ?7 M4 E6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells3 [7 L0 y4 s5 y7 v( m
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
) F) c/ ~/ u% m+ Z11. Chapter 9 Cell Adhesion/ k7 x  s. D% ^3 J
1. Introduction9 e7 W; X+ M5 F! [  W
2. Unit 9.1 Cell-Substrate Adhesion Assays
8 n" U9 z( R9 P* ]3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force4 f! {/ [2 N& ^3 }- z
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
" w# y" d9 A6 u5 d" x5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
/ V. r9 _* G5 S5 L8 R6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
9 W6 u4 L. ]- c6 _7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
8 X" J; H/ C$ o5 H, i" \- m# C12. Chapter 10 Extracellular Matrix, N: V" g8 o( R& X
1. Introduction
. @  }: x5 o- |' w2. Unit 10.1 Overview of Extracellular Matrix
  o. W8 |$ B7 m9 k6 |0 C3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
9 ~5 L; B% J  ^( \7 w4. Unit 10.3 Preparation of Gelled Substrates( J* {4 B& c- V1 ]7 \; Q
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9, o! h/ O# M7 W
Endodermal Cells; t) E# g: p# h7 a% s$ Y
6. Unit 10.5 Purification of Fibronectin3 V- z- E: P4 _6 B( v- M
7. Unit 10.6 Purification of Vitronectin$ r# B: Z2 W* F7 A
8. Unit 10.7 Proteoglycan Isolation and Analysis0 o: l3 C3 f" V  e
9. Unit 10.8 Matrix Metalloproteinases
% Q9 a# @% f0 a" ]  W6 ^' ?! @10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts' \; ]0 w: o( r; t
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
2 P# @. Q# n2 l12. Unit 10.11 Purification of SPARC/Osteonectin
# r" t0 j& I6 ]. U: ]: t4 z6 J& _0 b$ x13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
$ g: n- R7 u0 r8 \, U6 n; O14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
0 j8 e3 A/ [' y$ P15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
4 ^( G4 x" v. k* @( t( FXenografts
0 j3 n" J8 V! J- V3 |3 S  \8 L* l16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
$ M) K0 p6 S8 d# p17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
5 ~. @  q: h/ o0 _18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
1 ?5 _! G$ f% ?' E2 o2 D' s9 cCell Migration3 x7 z( y" S  L0 o
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
( ^" z$ z0 Y( U/ O4 }( W( P5 w' i13. Chapter 11 In Vitro Reconstitution
' W" s" {7 N2 f1. Introduction' h, e% l) _: J+ {; D8 q, M2 F( }. X9 S
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
9 u0 C6 F7 g  C4 K6 Q4 Y9 L2 q# _3. Unit 11.2 In Vitro Translation
% y, [0 p# n( \' t" G3 B4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells- s) G/ Y6 K8 X" v# B6 B$ ]
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
+ q+ Y, z+ W9 a6 n0 d6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
9 k: i8 A7 w, R" J- _2 e7. Unit 11.6 In Vitro Transcription1 Y  j! \- h9 N7 |+ L
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells7 p8 I- _. A# D1 l7 I1 {
9. Unit 11.8 In Vitro Translation Using HeLa Extract
7 U- A( z, ^  F5 t( C5 V. o10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems0 T" T4 J8 ~( z8 S+ Y- C
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts3 n3 O$ ~8 o; Q7 a" @6 T0 r8 O
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
% W, L" c0 Q& u5 ^! m: s$ ~13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
9 f" Q5 N% o% [6 v$ t3 ]14. Unit 11.13 Mitotic Spindle Assembly In Vitro
5 \/ ]  G6 T9 b% g2 f. p. [% A15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes4 y! x! U3 r. U/ K
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
; F# U. {6 V7 `; B- D  K! Q17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
& ^" X7 Q! I8 r- U7 b( S6 a18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts  q$ J1 a! M! m1 t# n
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
6 g* \* U' w; r+ J+ `/ H20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import7 e2 |" Y7 W# c' [, u4 g2 K/ \8 }5 u
14. Chapter 12 Cell Motility
9 i3 n# e9 O4 Q- p1. Introduction) C$ [0 \6 m8 l2 A1 L1 _! C8 G3 B1 {. e
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
, T2 B: W+ _. M3 t/ @# b7 n3. Unit 12.2 Invasion Assays
! G& ^$ T1 k4 M3 B5 O4. Unit 12.3 Cell Traction
: n; T& H" I5 z; ^2 F5. Unit 12.4 Cell Wound Assays
6 W/ @% b/ ?( N. z6. Unit 12.5 Dictyostelium Cell Dynamics+ O3 _$ x% H$ d. V2 E- z
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils4 p4 l, ~8 p- t
8. Unit 12.7 Actin-Based Motility Assay
# v; J' ~; C# d9 x8 \9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP+ ]7 [3 P* v. H$ c( s
15. Chapter 13 Organelle Motility
4 B9 H$ }! H% y" r1 n% A, z: R1. Introduction
' ?+ m4 O! x/ m  r1 ^2. Unit 13.1 Microtubule/Organelle Motility Assays
( q) o1 ?3 O. D3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin6 J9 G6 F# ]* a8 m' B$ J: g' l" ]
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP- W4 w# I5 p* |; l
5. Unit 13.4 Movement of Nuclei: ]( r4 A% B4 n+ K3 R1 H+ V
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
7 A8 k2 M4 R+ D  W7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly" ?  W% Q- J5 m( H% y
16. Chapter 14 Signal Transduction: Protein Phosphorylation; r# Z6 f. o& `, y
1. Introduction
" I) I8 Y4 n* L# S  F1 A2. Unit 14.1 Overview of Protein Phosphorylation: R' ^7 o) C0 ^. T6 |% H$ C
3. Unit 14.2 Immunological Detection of Phosphorylation
4 l- c( X& z% |4. Unit 14.3 The Detection of MAPK Signaling
& {, Q$ H! r+ _0 r* ^4 A5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation$ Z+ ~* i' d5 K' \1 l. w
6. Unit 14.5 Phosphoamino Acid Analysis4 s# ^( r, E: |4 `4 y, \- p
7. Unit 14.6 Determination of Akt/PKB Signaling
& ^  O9 r2 @1 [7 V8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
5 B" }  k, K( Y7 a* l9. Unit 14.8 Rho GTPase Activation Assays
3 v' h# O: b9 N' P" {10. Unit 14.9 In Vitro GEF and GAP Assays
& @2 ?+ W( ?0 j; |) X11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance$ f' [6 w9 I# Q' l0 l
Energy Transfer (FRET)* W& t3 R5 ^+ _/ c$ {2 r
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
+ \8 W. V, q6 Z- |$ c13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells) I  M/ ?8 x. b2 ]; W
17. Chapter 15 Protein Trafficking
6 Z! y2 T7 W+ C* y/ U1. Introduction8 C! z/ O1 j' x% B
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
7 h1 L4 K7 r, w! S$ N) z$ ?7 L  |7 ?3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking" t) W& K+ a: A: r, V
4. Unit 15.3 Endocytosis: Biochemical Analyses
& |& q% u1 {/ S; h- K, R( [7 T5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
0 x5 X2 k6 X6 _& R6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
7 H  G& n) U$ r8 _7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
! P+ X6 v+ K. r0 y5 H8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation. `9 T' p% g9 ~1 t0 h
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
! A1 W3 a" ~" n2 S! S% w2 r10. Unit 15.9 Studies of the Ubiquitin Proteasome System
; p/ \& v( a# l6 b+ g  e0 V& E11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
6 w/ c3 n2 V. i2 G/ G  b12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules: ]; n1 g" x' p  P+ F
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
8 g. K) q; P( V. o; P6 m+ k- m; q14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging. W' u7 e( e7 ^" s
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and5 ~' y% v! _& t9 \
EGF Receptor6 y% G9 r/ u" D: ]9 s4 W9 A
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers9 W' }, a) N  w3 t
18. Chapter 16 Antibodies as Cell Biological Tools
$ b* |$ R, V$ z, t/ g, i" Z! R1 T1. Introduction7 Y& L, k2 M1 A6 Y1 y+ B+ C
2. Unit 16.1 Production of Monoclonal Antibodies
3 \3 ?: M. A/ Q3 u3. Unit 16.2 Production of Polyclonal Antisera
; a: ~6 B% P3 R- p; k: j4. Unit 16.3 Purification of Immunoglobulin G! m  s/ r; B7 F' B1 N1 x3 Q
5. Unit 16.4 Fragmentation of Immunoglobulin G! b2 q/ f, \8 m0 v" `1 W& H
6. Unit 16.5 Antibody Conjugates for Cell Biology
6 w; c" ]5 {6 E, p( ^7 e9 @7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
  b8 d+ e8 X) X19. Chapter 17 Macromolecular Interactions in Cells
" _* ]$ |2 K8 T7 o7 w) K4 ]1. Introduction2 x: Q" Q; U/ D. }  g0 b* n! ~
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
  @& d  I/ C6 DMicroscopy
1 L5 x/ f, j: x8 {; a3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis- X, _- }/ u5 |) |  f
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
+ k- ~8 W# u4 U- _9 s3 I6 C1 T/ A5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
% f6 ^$ n& Z2 Z) e/ r6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
3 T: J# \; Y3 L7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors: x- g- ?; V% c
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
( j3 E! g( G' m1 h. QGenomic Sequences In Vivo- W. x* L2 T! l
9. Unit 17.8 Isothermal Titration Calorimetry
4 U) @" ~" j" X10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells) K- Q0 r; t# I4 y- B
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
: v/ N. c' M& E! a  L+ u! t* l7 J12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
. }' _1 w  E5 J' bInteractions (RFAP) in Live Bacterial Cells1 o! i$ Z: b5 g8 R  Q. t
20. Chapter 18 Cellular Aging and Death
1 p, ?9 G7 h( O$ k' w1. Introduction
, r( J' C% q) U/ ?' n2. Unit 18.1 Current Concepts in Cell Death
8 B8 |* n% @* V5 O2 `! e2 k% A3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
- O! y/ {# V/ A3 v0 _  t+ j4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria1 j. `6 z$ }& ]( h: r4 m, X: R
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
! ]3 S6 Q7 {. N6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death3 m. [( @+ ]) |& |2 Q, J7 i
7. Unit 18.6 Analysis of Telomeres and Telomerase
! V% J7 z/ i2 W8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of* K8 h+ x8 l2 Z3 s: V8 u6 d
Poly(ADP-Ribose) Polymerase2 r; k6 u9 e  @4 J
9. Unit 18.8 Flow Cytometry of Apoptosis/ m% b! d9 c& s% e% Y" B, x' H
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase6 y8 d* t3 M! I/ ^0 T
Assay
0 C5 Q& i0 V5 v( A4 i: M11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis+ q3 w6 n$ F' z! S2 F6 H! R/ @
21. Chapter 19 Whole Organism and Tissue Analysis
$ p1 r5 [  Q0 {3 ~3 d3 B( w1. Introduction* a! a' M% L0 b% f7 e& L
2. Unit 19.1 Overview of Metastasis Assays: r6 x8 H! D" T3 g) e2 t- G
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis! F/ z9 E6 Y- v. O) r6 L
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene6 R; y7 [9 J) S1 s5 A- J3 }5 o( _9 q
Expression After High-Fidelity T7-Based RNA Amplification+ k( ]4 ]- a. C# z, ?
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
# c1 l! l. Q4 F- o. F6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
' G/ N& {* H$ `/ g$ G7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo: Y" c" R- v1 G
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo% Y7 u2 K& x% d0 }5 T
9. Unit 19.8 Embryonic Organ Culture0 L/ b6 ^' f3 o  i4 g. ?' e
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
/ Q. Q; g; ]2 `11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
' t# Y  C/ Y9 o% a* y+ j12. Unit 19.11 Generation of Transgenic Mice( y4 j  X4 @$ M
13. Unit 19.12 Overview: Generation of Gene Knockout Mice8 H" F: C! M, f
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production1 Y0 X3 Z$ N) I
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection) O7 u1 c& Q& w  F! h) }
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
; X) r  L1 i% Z6 d1 p' L1. Introduction. [! z& D, @2 y9 m
2. Unit 20.1 Direct Introduction of Molecules into Cells
9 F: Z$ e8 R5 m) t2 ^3 P3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System8 a8 Q' d( s4 W. Q& {2 J& O
4. Unit 20.3 Calcium Phosphate Transfection
  S& y4 [( ]  X$ F5. Unit 20.4 Transfection Using DEAE-Dextran6 T- s  k% I8 O
6. Unit 20.5 Transfection by Electroporation
! S2 i6 e/ `, t" y  a7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
, R$ S3 {7 B0 A& p9 t* R8. Unit 20.7 Optimization of Transfection) R0 L! u- d% u+ c3 Q' J) C
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
# v, D' K" u* j- H' n23. Chapter 21 Fluorescent Protein Technology
+ V; x5 W2 P/ _' f' T7 Y) L1. Introduction" f4 ^, S6 I: \2 h8 J" `* u
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells# g. \7 R0 v: Z% n5 a5 P
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)4 `' s- S. D$ Z
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence5 V7 z  ?7 s: K2 c' n
Complementation (BiFC) Analysis
2 y! Z: M7 d1 {. f5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
( c$ p1 y0 R% F3 Q+ F6. Unit 21.5 The Fluorescent Protein Color Palette
% F' n% q* ~, }! Y! u% ~: ^1 _7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
, C5 i; I1 p  z4 ^; L24. Chapter 22 Cell Biology of Chromosomes and Nuclei
' y3 T. k) W4 G) c$ s1 N1. Introduction5 {7 C- b9 }, B0 L" I3 w( o
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
5 {0 w1 ^) T9 u) i1 E  n$ h3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
* J) e8 W& y7 E) y: J) I4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis- B; j; j& G: U9 }6 n1 N; o+ g
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)2 t  i. E. v! [+ h6 H/ ^
6. Unit 22.5 Multi-Color FISH Techniques
9 p$ `% N! t$ C. \2 Y5 ?1 t: Z) o% h7. Unit 22.6 Comparative Genomic Hybridization8 H7 w9 A% y- F' ]
8. Unit 22.7 Sister Chromatid Exchange
) ~/ T" Y6 o, A5 B& E' c' A2 N! S9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
9 {7 X; y1 {  O10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes( `" k' t' o8 d0 m. N! C6 w  N
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
6 L" Y" S1 D% m' p5 x6 _9 y9 ~12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
: o, X$ ]' [2 I0 g  \4 }# H13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells6 j% i- q8 f/ `* k: @' R, {/ `4 F
14. Unit 22.13 Monitoring mRNA Export
% l9 a) N* I' e% d0 j15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-# j! {6 C0 l4 ^- Z+ l+ Z) t4 b
Field Gel Electrophoresis$ B  V; w. p8 i7 h( K" Y+ K( O  H6 f
25. Chapter 23 Stem Cells
, N5 |3 R: u0 |1. Introduction
3 p" r/ R) T7 u5 X0 J# Q2. Unit 23.1 Stem Cells: An Overview
3 G/ A# ~1 }8 a, W) ]- ]9 [- W3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture( U4 n. G" p; o& [2 Z
and Differentiation as Embryoid Bodies* U8 I$ B& v$ z, |' I4 n- V
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood# P; V. E1 _; _0 e. E$ i3 f, z
Vessels
# d2 v8 Y6 y0 Y4 ]" J% V5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into* {- X, W. ^( Z
Adipocytes! J0 p7 h) K+ }+ }# c4 k, M' M4 v
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation  a9 \' ]; ^- u( L* d8 E: |
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
7 v# t# I& g0 D8. Unit 23.7 Neural Differentiation of Human ES Cells3 v5 l: A! p. F4 ^
26. Chapter 24 Lipids
; T* n* L8 `+ j' E1. Introduction8 T7 |% l$ ^( R2 s  A
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
/ B( k7 z) ^0 e3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins8 k6 @* _6 t% m9 J$ ?2 j% h! a
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
4 v6 L# p0 `+ e$ Q5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains3 I' l& h) C" P
27. Chapter 25 Nanotechnology
& U- B+ Q; @/ B/ m; T: N1. Introduction
+ t* D* F( l2 n2 I! l" k4 a2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes% d, V3 b, U' P8 Z! J
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering% a$ o* x2 ]$ s/ |7 [
28. Chapter 26 Viruses
/ k# C+ X5 \; l1 [4 B1. Introduction
  B5 C) I% D2 E( L6 u2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
6 _, A7 v# }$ A/ r/ S* \3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
4 R2 ^0 E3 V8 \Cell Entry! v5 k- m/ C5 S, f9 m: `5 F
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
& N' D" C1 e# X8 j4 f1 C0 k5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses: K3 Y8 @: p% g
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission+ `! I  w  X" p
29. Chapter 26 Lipids
, v8 }2 {5 @( d1 {5 h2 B" h, S% I1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
! q/ U# M5 v0 \1 j$ \' OReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
9 q. U4 T4 l( A: `( n- Q3 q; n2 ~( z30. Chapter 27 RNA-Based Methods in Cell Biology9 D1 v; K1 F$ Y7 i: Y5 R
1. Introduction3 K4 `8 H' s/ q8 ^$ f4 D& z
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
* o7 @- z7 X' D7 q4 F7 o3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
2 N+ {, F. [! ]* x5 k" r: QRescue in Mammalian Cells2 o9 Y* Y; @7 E! \1 @' [
31. Appendix 1 Useful Information and Data
) a/ k1 ^' }; v+ _1. 1A Useful Measurements and Data
) a! L9 M# Y* y8 ?' D0 k2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
/ `5 Q, N# X2 `. N! C3. 1C Identification of Motifs in Protein Sequences. G. m$ o. \- _3 M; R4 n
4. 1D Safe Use of Radioisotopes- ~! p& Q6 _# I2 H9 b' s% b
5. 1E Absorption and Emission Maxima for Common Fluorophores
: Q3 M0 L: H# o8 O4 e  H6. 1F Importing Biological Materials
2 b* M+ k9 o8 ~  n+ ?7. 1G Centrifuges and Rotors; z$ J' ~4 b4 }) b2 f# x
8. 1H Internet Basics for Biologists: P; S+ i4 @* \1 r
32. Appendix 2 Laboratory Stock Solutions and Equipment
8 d+ ~- d* Y2 b- P5 a: L1. 2A Common Stock Solutions, Buffers, and Media
# x; Z: P; b/ A2. 2B Medium Formulations" Z; o! S/ `  J0 ^
3. 2C Standard Laboratory Equipment
7 `* ?' e. t3 ]+ K3 S" v8 v1 ~33. Appendix 3 Commonly Used Techniques1 P& k6 _2 e: D$ E& ?
1. 3A Molecular Biology Techniques
. g2 k! k3 G/ D2. 3B Spectrophotometric Determination of Protein Concentration% d9 P, b# q  w9 b$ b  c& |
3. 3C Dialysis and Concentration of Protein Solutions: {8 Y, `9 `. E( `
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
" z2 K3 ], _/ |6 W5. 3E Silanizing Glassware3 `4 z) v" Z3 O9 d
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
$ i) b$ n0 m; j- C7. 3G Micro RT-PCR
( {# d* m( o, U6 s) T: `8. 3H The Colorimetric Detection and Quantitation of Total Protein3 _1 V( q; h! i3 l, o& |
34. Appendix Suppliers  \: @6 B' u) p2 f. u& d7 G' T0 I
1. Selected Suppliers of Reagents and Equipment
" a; U* b. V5 }
! `. D/ |; }9 ^- @6 r4 X% i; S

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