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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
' L2 N: K! G8 n# p
: j7 i# a' Y; X% J- vOnline ISBN: 9780471143031
0 F! F. M0 V S; w0 xDOI: 10.1002/04711430303 h" v0 D/ x; ^6 U7 ? E0 Z! Z3 ]
, n1 ]( _& z% |% L0 r# C% eTable of Contents
2 T8 W! q+ Y: B1. Preface9 U- j& ]2 n2 |/ p+ c+ {) I
2. Foreword
! G$ `# ~9 U7 e3. Chapter 1 Cell Culture; b8 k: L* W2 n- S
1. Introduction3 E. t6 E- P0 h' T% X) Z
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
7 Q$ Z5 e7 k& M" u1 x! }4 ~6 `3. Unit 1.2 Media for Culture of Mammalian Cells1 D- F- N2 ~) c; ]
4. Unit 1.3 Aseptic Technique for Cell Culture
9 Z7 q3 b8 ^4 R3 ~4 |; F% V+ S C5. Unit 1.4 Sterilization and Filtration! I3 k9 ^; C$ y7 H. G( H
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
( h, }! [ Z( n5 @" ?7. Unit 1.6 Media and Culture of Yeast4 r9 S( F8 c( r
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
4 [- z$ x" `; _/ l4. Chapter 2 Preparation and Isolation of Cells5 r: q% W# R. M0 O- f6 k; [ I
1. Introduction
. a8 Z# j/ w; s1 g2. Unit 2.1 Establishment of Fibroblast Cultures1 M/ t, M* e' L8 v. g6 g3 P
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
+ J i/ q) g1 ]1 g5 j6 V8 o4. Unit 2.3 Preparation of Endothelial Cells3 g* W0 ]/ s7 @8 q) A `
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
1 H! k: R7 y" H4 [3 c6. Unit 2.5 Laser Capture Microdissection' U) o, t$ s+ m# O* C
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
. C# q2 O9 w$ k* G8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
$ y+ e- o! t0 V7 l4 c. C) B7 q5. Chapter 3 Subcellular Fractionation and Isolation of Organelles+ \& L9 a" O1 G" Y9 S
1. Introduction
6 K1 ?. R/ {& h8 I( \9 a2. Introduction
/ W9 Y7 g0 z5 K& A3. Unit 3.1 Overview of Cell Fractionation
0 @9 q# q; }1 |% \3 d4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
6 f1 K* g) V' H" J* ` {; s5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
3 k5 `4 S0 v7 ^2 R4 G0 X9 I6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
$ ~9 d3 B i- u9 Z" [3 Q7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient3 K& ?" O0 [5 o& m+ R
Centrifugation p' o2 Q. D- s
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
" @/ [7 z+ x: ~. G0 S4 bCentrifugation: j5 S! U5 g- `9 o: ~' H
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
- i( ~: k) V p/ L! l10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae$ ?9 {; _; E L5 P% e
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient; m( g# d0 i3 B
Centrifugation
. Q6 A) ~2 k% B/ q; a$ S8 [12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues8 a: U+ l9 B$ [8 `: U
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
- Q& s# w0 T2 r( ~14. Unit 3.12 Isolation of Synaptic Vesicles6 A- P$ L* o# M# N O3 S, t# ` }
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
5 v y4 e- c" A* m- {: K3 c% n& a16. Unit 3.14 Isolation of Melanosomes! ?; L5 k _7 p K$ }" d
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation# ]& |: L+ r. w4 J# i* o* h, n
18. Unit 3.16 Isolation of Mast Cell Granules
4 H; I* l& a; z( X6 @" ~0 i19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster9 o& c: S8 [+ @: W' T
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas# { [1 \3 Q# M k: {
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
1 C T: p& H" E1 {2 H+ ?) w6 c* ~# i22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
- x3 L3 m- l8 z5 X23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
6 W6 [5 E1 e5 T3 E8 ]' ?$ i24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
- b5 H$ k) C# c4 qFluids5 t* b( @7 Z1 n1 b% Q# {5 ]8 [
25. Unit 3.23 Isolation of Intermediate Filaments
( k2 |7 R; Y' B3 Z O4 S3 ^/ D26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle G( ]9 g/ e' Z
27. Unit 3.25 Isolation of Myelin: f, n+ j, x( h5 c6 A( w, {+ A* s
28. Unit 3.26 Isolation of Renal Brush Borders; ~4 j! x# e; s$ J" p) P
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane2 o# W- n: M/ i9 v7 X) c
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts6 I% e, l' M5 J1 f& t! f7 G7 f
30. Unit 3.28 Isolation of Amyloplasts% |% e* z: t9 O1 i* G; q
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
' {' k- a$ Q- z- N8 n3 ^# Z+ f( j& k32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic% h3 y- w& }% h! R
Centrifugation
1 ]& u) O3 n9 q8 M# J: L% G9 s33. Unit 3.31 Isolation of Neuromelanin Granules: r' U3 `. x$ p2 X
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and7 f6 Y' e- D5 E
Density Gradient Centrifugation) Z: h) O- o( [3 R
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
& o* c. S3 {# b0 Y' m% g36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation k6 T& g$ u( \, A
37. Unit 3.35 Isolation of Platelet Granules, ?8 S8 N' Q8 S9 Z3 M3 \# ]- F. H6 M
38. Unit 3.36 Isolation of Nucleoli
' A* N0 @! Q8 K; W39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
' [: b1 E1 i7 c, z# L, f+ j1 s0 X40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates+ P) F2 f9 F- F" q7 Q
41. Unit 3.39 Isolation of Chromaffin Granules
/ z/ Q. a6 M# s j* v& C* W5 T42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
/ q/ @& ?" a& t* j$ \2 P" D1 A, N) U6. Chapter 4 Microscopy, \' `4 h, ?0 ?! D
1. Introduction2 [0 Z- D" j/ r. C Y/ _+ ~
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope6 j* v2 h e; s7 q d* k
3. Unit 4.2 Fluorescence Microscopy
4 w) X+ q' ]' j# z7 \* A( Z4. Unit 4.3 Immunofluorescence Staining; {/ r& |( s( x$ F9 a
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
# c: b, \7 _# A t/ `+ |6 i6 o" g6. Unit 4.5 Basic Confocal Microscopy( W+ A* ?; |- E7 B* }' F/ ]! q
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues. b1 @4 S5 J- l
8. Unit 4.7 Cryo-Immunogold Electron Microscopy
, N C6 K) k2 u+ s% _9. Unit 4.8 Correlative Video Light/Electron Microscopy
4 R' ^! o# U6 |/ ^2 o& }7 d10. Unit 4.9 Polarization Microscopy
0 J# a2 A: c- U11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
# m* n/ u; K. T3 L# C12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues& [3 |! ?! X. F7 D7 o' `' ]3 K
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface8 D5 E; `, e2 c) M- G A4 `4 ]
Events7 e J @2 o) \" f
14. Unit 4.13 Fluorescent Labeling of Yeast
# @9 L. K) |5 Y% P5 f" ?6 C15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy+ |1 w1 t: u/ Q" Q8 i7 h0 C( b
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy; y5 R% j1 @& T/ e+ E! s% b
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
; S: _+ E/ e+ gin Endocytosis/ L _, x1 v* u$ w/ w
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology) D7 u5 [8 e" S) h7 p3 R- D3 A
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development# g# y/ J* A6 C; r7 h
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images) L. V7 y+ I, }6 q
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions4 _- T# [! u5 d$ r- t$ w% [# d
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
! f1 v. t: d+ k' k23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
! A( c8 ?; I6 n/ ]- F24. Unit 4.23 Interference Reflection Microscopy8 i& B) P% k. {; f% v C. D
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
: p; N2 R$ Z9 a, s5 _/ e- I; E26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches2 i7 @; U+ l; P+ M1 r9 I4 K! ^8 G
27. Unit 4A Organelle Atlas: Appendix to Chapter 48 Z7 z0 S q7 ~. Y' `
7. Chapter 5 Characterization of Cellular Proteins9 d3 Q3 R! J. J; }! i- P8 j
1. Introduction/ ?# Z7 T1 x! J! F6 G3 z
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
; v) c% c+ |& a4 b, I& ~ q3. Unit 5.2 Determining the Topology of an Integral Membrane Protein' J* m$ E; k. O9 |3 s$ a
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients, s( [) F1 ?( Y. U" [# ~9 v
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
3 r5 A! O; J9 y- A6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
# f! K+ F Q5 b5 F% X6 y* n* X7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
- ^% \) p/ J4 c8 Y* E% y9 G nSpectrometry# h6 t) n' w; K6 Y
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
8 a& K" N4 q, nApplications: T" E( J3 J) F/ o1 R' h
8. Chapter 6 Electrophoresis and Immunoblotting7 \+ Z! f9 ^9 i& J" ~9 N/ ?$ G# A
1. Introduction1 E5 [. w s1 G) C' L$ W+ N3 z
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins) G- P. {6 ]/ o' D
3. Unit 6.2 Immunoblotting and Immunodetection
+ |3 a; J/ x3 E4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
7 s& [, _2 F. h3 ^% X5. Unit 6.4 Two-Dimensional Gel Electrophoresis
; w6 G' s: g$ c8 _$ X6 Z8 q+ V) b2 g6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions* s/ Y$ |2 @, }- G
7. Unit 6.6 Staining Proteins in Gels
* [7 v; E2 P" p( ^8. Unit 6.7 Agarose Gel Electrophoresis of Proteins5 J0 b# G7 O) x+ g- _+ q3 [
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
7 x3 s" X' U/ q5 M4 ~2 [9 P10. Unit 6.9 Digital Electrophoresis Analysis: S- w4 B9 W+ e8 k2 ~/ r3 i
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis. R( H" T0 y+ b' r/ h M) v3 M
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of! S7 o- o. \: |2 x3 s
Single Cell Gel Electrophoresis (Comet Assay)
+ B8 z& E1 m+ P' j4 v! m9. Chapter 7 Protein Labeling and Immunoprecipitation! P+ X! Y& H3 Y& Q
1. Introduction. ]3 h- p% B! l4 ^5 W4 B6 Z( Z
2. Unit 7.1 Metabolic Labeling with Amino Acids
2 x+ K1 f+ T! D0 z7 z! }' h3. Unit 7.2 Immunoprecipitation" {! r, Q# z" y+ e: g% h% _" K; ?
4. Unit 7.3 Metabolic Labeling with Sulfate
4 {' Y6 i- e. U3 H+ w& s# u* B& A5. Unit 7.4 Metabolic Labeling with Fatty Acids" |' l4 Z" i+ g- D
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups" d+ ~$ D5 m0 z% x3 |; q
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins6 f0 z ?) j) o/ }" M3 _. I
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
; M+ d4 J8 @3 J& y( M9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
- z. Q! H+ {; B- ~- B5 `% ~% y: p10. Unit 7.9 Analysis of Oxidative Modification of Proteins
6 y6 P' B ]: _* q7 v3 w11. Unit 7.10 Radioiodination of Cellular Proteins
8 ]( P: j8 Q4 Q6 F* c: q- q. m* g7 r10. Chapter 8 Cell Cycle Analysis3 O3 ?. @0 k) E8 j
1. Introduction8 p4 b0 ~5 n" _# `, j8 h) U
2. Unit 8.1 Overview of the Cell Cycle! P8 N; ]: l f# o
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle5 d+ o# _' }6 m7 m* U/ X% {
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
- O1 N T& G8 G V5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
5 |! O7 n3 t$ r: ?. g1 g6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells- K, N6 Z# I: u1 O
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
% l. u* [! m1 j8 _5 k11. Chapter 9 Cell Adhesion
/ J7 q) N, u$ j2 k+ L1. Introduction
# s2 O. q9 ]3 U. \2. Unit 9.1 Cell-Substrate Adhesion Assays
' j5 p; W8 S( V+ h; }% b0 C6 N$ P3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
* z5 p9 t$ U* p6 {2 g4 M4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
; Y# w% p# ]$ w0 z5 e* O5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
4 D4 Q, h( r8 c' g4 `" k9 C& ^4 T6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
2 L% D6 c7 G; @7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
7 s: b% b, {% Q" `; W( Q0 X& ^12. Chapter 10 Extracellular Matrix
4 d7 r& x. e. q: x: }$ k" i* D1. Introduction0 w, \$ @/ S! W
2. Unit 10.1 Overview of Extracellular Matrix
1 B4 ` H+ G1 |7 Y, _3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors6 `( B* X( J9 X
4. Unit 10.3 Preparation of Gelled Substrates+ B, \ Z, n$ G/ F: C
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9+ ^ L, }: H& g7 Z1 M
Endodermal Cells9 s7 _- ^$ H" X; O- _8 @# i& L
6. Unit 10.5 Purification of Fibronectin
* {5 p K! d* z" Y% D( B7. Unit 10.6 Purification of Vitronectin
$ r4 g$ u$ [! r0 {8. Unit 10.7 Proteoglycan Isolation and Analysis" e- e9 J3 P/ Z$ e0 H6 P# _. Z
9. Unit 10.8 Matrix Metalloproteinases
3 _. `: C* a5 }4 ^! r10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
t/ z) Q: y0 G8 p11. Unit 10.10 Purification and Analysis of Thrombospondin-1: P: W6 w# H2 M1 G
12. Unit 10.11 Purification of SPARC/Osteonectin
' A* Q: N- T2 c/ ~) K1 A7 w% T j13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
8 B9 v. r5 G- u1 s14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly9 \! D! H( H, V% U
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor2 z' G% t" o& U t0 e) Q8 p
Xenografts* ]6 _0 w- s0 J* V3 E; y) z- H
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
- @3 |5 I+ V8 P9 l7 W3 R% I17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties/ o, R, U9 R: \* m
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D3 a) Q3 \ t& q6 V
Cell Migration& U+ Z& Q7 G7 k1 Y2 X
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
W* V g' q% z- l3 s7 E13. Chapter 11 In Vitro Reconstitution5 L: Y$ o: T. b( f
1. Introduction
+ a4 ?. u1 O5 B2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems/ g3 R% Y: j8 B' k: j* q
3. Unit 11.2 In Vitro Translation
Z/ m0 q5 r# K; t1 B" P# b4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
! [8 H2 T0 `# G* n7 ~" F! N& e5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
; {& W6 u* e$ ~% o, h2 t& }- R6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication' s( V; D. c9 r- f- D- ^
7. Unit 11.6 In Vitro Transcription
. e a/ }% e; E# {$ Z3 o( o8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells0 p: o; h* e q: L& S
9. Unit 11.8 In Vitro Translation Using HeLa Extract
: w% E7 w$ o* A# {0 r6 E10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
* ^1 l2 {6 z- `11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
* X( ]" J; r" S/ f12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts) F9 _+ L$ _( f1 Y% T; y# ~
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
I6 c6 J' f8 K14. Unit 11.13 Mitotic Spindle Assembly In Vitro5 V7 O- s4 s: T4 ^& Q. J. T* [
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes# w# \ u% y, E2 B
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import+ Z7 p5 v$ {/ k) J. [+ D
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import( a$ ~/ [# K5 O
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
" y; d% R3 G/ p m9 g5 T' n# r19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells/ }: K3 w6 D5 h( Q3 E9 M* X) s7 J
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import2 \+ |/ l) q) t" A3 K
14. Chapter 12 Cell Motility: V4 k K& W- e9 ~/ H
1. Introduction q3 w3 `- }9 [0 l( w
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
, _( ?" M0 O' a7 D7 n$ h( l! d' q3. Unit 12.2 Invasion Assays4 @/ e4 \* W5 V0 z/ O
4. Unit 12.3 Cell Traction
8 I0 y3 a: c! h4 g- R5. Unit 12.4 Cell Wound Assays
. b, w1 k. v: c B6. Unit 12.5 Dictyostelium Cell Dynamics5 m) n$ n {6 U! a- I/ }
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils( i) g5 D ^( N, j' W" a6 e0 @
8. Unit 12.7 Actin-Based Motility Assay0 t9 n [0 U% d# L0 u/ N# K
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
# ^( | t8 F& c; e- _6 G: ^7 y% K15. Chapter 13 Organelle Motility
. n8 [* Q" R. H$ j7 K1. Introduction' w; b: `8 C3 ` L; ]! ^. g
2. Unit 13.1 Microtubule/Organelle Motility Assays* L5 M5 _/ n5 B& r# @- ?" i
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
( l. z" [) z; e9 n( y2 A4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
; o5 g0 z3 A" B1 |0 D+ {3 m: h5. Unit 13.4 Movement of Nuclei+ y K& G7 \0 i; L
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching. |- d! _0 O; q) V
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly; d, o" T7 X1 H& ~7 X: k# }
16. Chapter 14 Signal Transduction: Protein Phosphorylation1 f' j w6 |4 Z5 l) j+ p! }! _
1. Introduction
1 g7 a$ u/ F( w) n; ~2. Unit 14.1 Overview of Protein Phosphorylation
0 }2 e: }1 |' U4 u! {+ {# `3. Unit 14.2 Immunological Detection of Phosphorylation7 l1 c6 @; q0 t
4. Unit 14.3 The Detection of MAPK Signaling
. |( I8 F6 C1 }* }' a, k5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
; N) _1 m3 O9 f% F; D6 E* q6. Unit 14.5 Phosphoamino Acid Analysis
( x- @2 i2 n5 z$ k5 \, u7. Unit 14.6 Determination of Akt/PKB Signaling
) O. n- G4 p+ X, s, v F' d5 F8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events& A$ @. o- y0 C0 m( t* c* N: |
9. Unit 14.8 Rho GTPase Activation Assays/ i' c6 r1 }: ^/ Y) \9 S4 L6 A* a8 p
10. Unit 14.9 In Vitro GEF and GAP Assays
# ^2 x* G/ |) u, u- b11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
' S) Z- v( r/ AEnergy Transfer (FRET)/ B: s; m: o1 W5 R
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
% ?; g! ^( ]1 i/ y% F13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells: `+ D" ^& S6 I' n4 g& {; Q
17. Chapter 15 Protein Trafficking
- |! E* n; y. P7 i' Q1. Introduction6 ^+ z$ g4 O7 G* v% n6 Y) L9 N
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways. P. e, h& [0 {2 Y" G1 B
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking9 n% i1 g; v' |
4. Unit 15.3 Endocytosis: Biochemical Analyses( J$ a' ?+ o; N8 L- G
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
6 a) G. Z" z. o; Z: N0 S- F6 U' `6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
. f9 t6 i( f' h+ N! z/ x$ q& [7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum. c1 F& c$ i# ]! w" l0 i" O
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation6 [1 V U A7 r9 x
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
3 b& R; h/ N/ e3 @0 s10. Unit 15.9 Studies of the Ubiquitin Proteasome System5 y& C+ Z d! r6 E) Y
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network5 ^( @" P" r% W7 ^+ X4 d
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
: ~* t$ g4 u/ M `# S! f: \13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells- N# R# X, B, I3 N; F# \3 X1 N
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging! g4 \0 ?. u) `8 ?6 f
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
# B+ @% z+ H+ v6 x/ p- Z4 YEGF Receptor/ ? H6 F% O* s
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
: @4 R4 T7 v" D$ O; Z& n18. Chapter 16 Antibodies as Cell Biological Tools1 p' r; f7 R" \8 n$ O3 C& A
1. Introduction
) V/ B" D9 n- O& [' O2. Unit 16.1 Production of Monoclonal Antibodies
$ o1 F0 W4 c3 [; J3 T3. Unit 16.2 Production of Polyclonal Antisera% g# d% T2 w* U4 y$ k
4. Unit 16.3 Purification of Immunoglobulin G
h; b t5 b: R& [: n0 c5. Unit 16.4 Fragmentation of Immunoglobulin G
% t* Y! a" @# m! [; ]1 ]6. Unit 16.5 Antibody Conjugates for Cell Biology* c% z. O2 y/ f# i" g
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides1 v/ D# C* T1 i
19. Chapter 17 Macromolecular Interactions in Cells0 x. D( c. t% ^- w9 T8 r
1. Introduction
" i; b4 p0 ~6 ]' U6 B" X( E# D2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
$ _8 I3 S8 I3 JMicroscopy) p% p8 ~; K$ X" a
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
, a& s0 R" f# ], e8 P4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
5 A) K/ A/ z6 H* ]8 T9 j+ J5 ?5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries7 l) s) a1 x4 ] n
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry0 m u3 g, @/ P: y8 h
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors7 \" O! u; u+ e8 ?
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific3 D- l! P; I& b' n
Genomic Sequences In Vivo
5 q, D7 D9 u% c0 C# U- |; l) L4 {9. Unit 17.8 Isothermal Titration Calorimetry
, L# k: F- [# F, V10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
0 H' m* I* I# G O11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
* O1 J! h+ E& y4 x$ A* f12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
3 K5 Q E) J" y- ?9 A$ W1 f* vInteractions (RFAP) in Live Bacterial Cells
+ k8 S' e% u; |20. Chapter 18 Cellular Aging and Death9 E3 y0 H( W! j
1. Introduction
& U3 u% A7 G% `) m) S! M' c2. Unit 18.1 Current Concepts in Cell Death* \4 Y6 ?1 I5 J% k, Y" H! e, ^7 f
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
6 n) ?* f! {0 [+ j0 E4 k4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
+ G" r* M3 Q: x7 \- X" v' \2 l5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH), u8 i" ~* @% m* j. t( c$ \: i! t
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
# `# Y5 N! R# _& h7 s. | D7. Unit 18.6 Analysis of Telomeres and Telomerase. G; ~) l- G- e
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
2 {1 u% {! }" c6 Q) v: G% a4 N" {Poly(ADP-Ribose) Polymerase
" {6 p- v! I/ `; i+ ~" [4 _. H9. Unit 18.8 Flow Cytometry of Apoptosis
& k) J" C/ j- p+ F- J# U8 u' J10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase0 ]. T! k3 @" H7 R/ a: b P* n$ o
Assay+ B& _3 N; K( A' x7 a1 s0 V
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
# B' G& \# W J0 F. A21. Chapter 19 Whole Organism and Tissue Analysis
5 s1 f# ~, D6 s1. Introduction; C f. ^7 |% x3 o
2. Unit 19.1 Overview of Metastasis Assays
5 ~0 Z6 s' U) ?1 V0 d4 m2 k3. Unit 19.2 Tail Vein Assay of Cancer Metastasis. ?* {) ^: F( ]. Y) N5 o: q
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene+ t; m: {; j4 n' v" O5 k
Expression After High-Fidelity T7-Based RNA Amplification& R4 n! z6 d, d$ M
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA6 Y% t; W0 q2 F& z" U9 Z. {
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model+ Z. [- L# p& X
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
9 l. a9 F6 i4 O7 a* D* n1 T% C* k. A8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
9 {3 p; l( b2 }! u. o8 ?9. Unit 19.8 Embryonic Organ Culture; Q. t) z, x$ N% `; _1 v0 I [
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin9 t) [/ h0 X- u; ?2 K, y7 w. }4 j
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice& P! u+ O7 V9 [+ G) j7 B& y9 @ X
12. Unit 19.11 Generation of Transgenic Mice4 {; |2 C. y/ q. E
13. Unit 19.12 Overview: Generation of Gene Knockout Mice% W. i4 a0 L7 z9 N; g m
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
, h( `5 H: a) |: e( b# A15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
1 f C% a; O( n, P6 L) T22. Chapter 20 Expression and Introduction of Macromolecules into Cells
$ Z, Z$ d* I P+ v1 \1 p) S( ~1. Introduction
. p& w4 ^2 Z% Z6 Y5 o& n2. Unit 20.1 Direct Introduction of Molecules into Cells
* X' L3 }/ Z ` l V3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System/ y* `6 `& h1 c) r
4. Unit 20.3 Calcium Phosphate Transfection
& I7 i/ y) Y* `6 S4 U; r5. Unit 20.4 Transfection Using DEAE-Dextran
# i* h: v+ M1 ~* E8 U9 [6. Unit 20.5 Transfection by Electroporation
& l; o( ^' ^& U- S2 B+ Q7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
( S Z* U& ~. M. }! n- _" ^8. Unit 20.7 Optimization of Transfection5 v+ y1 |! Z# {/ O8 Z: T
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System+ R- a3 x: h9 m3 O* y* T+ M
23. Chapter 21 Fluorescent Protein Technology
% v- c6 N \1 J" r% ~1. Introduction1 W2 {* V0 c) G
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells/ s- E1 E9 _- J
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
# R" g2 t6 H) {4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
+ Q! D$ k% |* ^9 O' {Complementation (BiFC) Analysis
6 G0 i2 o. x: V5 L# n; s5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
8 \2 K7 G0 W& e! w. O7 |& E6. Unit 21.5 The Fluorescent Protein Color Palette
8 u& R5 ?% A, n1 b/ l- {3 G7 ^7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
- ^7 v! s a) x0 b' G6 n, B2 f24. Chapter 22 Cell Biology of Chromosomes and Nuclei
8 y& C! {$ f2 Z0 Y" |1. Introduction4 B: u+ j6 }0 ]4 [1 X
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
' B! {/ n. a/ k$ d" K! `6 H3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
: N/ ?* U4 @# ^7 j; `6 t4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis/ t2 m& v. y2 J0 W# N% T) L5 {
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH). c8 m9 C4 _4 G* ~2 {
6. Unit 22.5 Multi-Color FISH Techniques
& ]0 N1 f9 W) z5 X7. Unit 22.6 Comparative Genomic Hybridization
T: D) L" G& t0 k5 X% I8. Unit 22.7 Sister Chromatid Exchange2 a ^" t" Z4 j+ h: Z
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
, N2 c$ ?, C/ I& _+ ~10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
4 f" ]3 R5 p* p; H( X/ t11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs, k1 l# L/ ?4 F, i% j K
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
- ?$ n) \9 b( }3 l- @13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells- i1 c% W& A! v# O& K
14. Unit 22.13 Monitoring mRNA Export" Q7 G0 l9 ^" c
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-6 w& ], K, {3 J5 ?, ^- ^7 s* s
Field Gel Electrophoresis! |6 R O- E+ A2 i8 x
25. Chapter 23 Stem Cells! F5 a& H$ N7 {! F
1. Introduction
/ P! K- x$ b- r! e5 ^2. Unit 23.1 Stem Cells: An Overview. ]- K A8 R! c. S! E$ [ Q
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture$ _- C& Y9 I. R
and Differentiation as Embryoid Bodies
8 c- X& Z) @5 K3 h* J8 L R4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
; |- f$ c `) u5 A# sVessels
& ~; m8 Q# E: V6 p5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into0 |# P. V% [# B, }" J
Adipocytes
, M& w; P, }" j' I6 U5 F6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
. c# l6 K+ n( z7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
- d. K8 H) u% K. }8. Unit 23.7 Neural Differentiation of Human ES Cells
7 Y G$ g) z" Q6 X6 B26. Chapter 24 Lipids$ n+ u% s' m$ @( z7 p% I6 k9 S
1. Introduction
' M3 \! O, b9 h. \& X+ l5 d# J2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
: f/ D- `+ a9 v& ]" k6 x) d3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
{& G* a. C; @( q, `, J/ P. g5 b4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
0 |/ V+ T8 T3 u$ r( S5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains+ E$ _. H& L$ \. Y; k* n
27. Chapter 25 Nanotechnology
( W9 h& r T# W; @$ E1. Introduction
/ x& D# H8 l+ f7 K) x2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
# H5 l& a& [. J5 ^. D: {4 Q3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
8 z3 ]8 O% D; J3 l" X, S& N28. Chapter 26 Viruses9 ?! i6 B& T& F3 M5 ~5 C, x( G
1. Introduction! ?/ L* I2 [2 \2 T. v6 ]' v
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
9 g* L8 b2 A7 N( z4 ~ G* X$ j3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of; A9 R; f$ H) q. u
Cell Entry
! D" s( n1 v4 P' d: e4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
1 n0 ?0 a* m7 v+ C& A. H+ g1 h5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
9 Y3 E: C3 o3 Z- B7 ]- s6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
1 t0 z/ |4 i% u: M29. Chapter 26 Lipids% O& ]! B4 F" H2 |) a5 ~- |/ i4 K
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by0 F5 ~5 @( Y3 \' \. C
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication* ?5 V( h) Y/ i
30. Chapter 27 RNA-Based Methods in Cell Biology
/ w: j+ V; k# H. e6 y# n- X- J1 Z1. Introduction* D# F/ }, {" k7 q0 o! u+ u# @
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
9 b5 q0 t: W( \; }3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible$ z4 F# Z1 d2 p' u# m
Rescue in Mammalian Cells
# `- k8 ^0 @/ G8 k: r a- S31. Appendix 1 Useful Information and Data2 Y/ T0 G% ^! Y _
1. 1A Useful Measurements and Data. ^- X( ]6 M' C i* U" d
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
' t1 ?7 h$ j! I% W# H3. 1C Identification of Motifs in Protein Sequences* f. \; S& s) k
4. 1D Safe Use of Radioisotopes
8 q% Y( l; P! ]& [' |5. 1E Absorption and Emission Maxima for Common Fluorophores# w! r5 a) p- q5 l$ s4 Z# w
6. 1F Importing Biological Materials4 p1 a) R: } Y' p" C1 @0 }, }
7. 1G Centrifuges and Rotors
1 y9 F: a% Q8 T) [' u3 T% h# S8. 1H Internet Basics for Biologists
& ?: \8 }" e# g& \32. Appendix 2 Laboratory Stock Solutions and Equipment
$ b5 g# p, z# m# p0 r1. 2A Common Stock Solutions, Buffers, and Media
; R+ O# \5 P+ q) j2. 2B Medium Formulations
. N* T/ `% m6 k/ G! Q3. 2C Standard Laboratory Equipment0 @8 L3 ]: @1 P! d& [) P2 e
33. Appendix 3 Commonly Used Techniques6 B/ M; l4 p1 D r: i8 m: s, r' {1 T
1. 3A Molecular Biology Techniques
+ U( X! y6 v& n2. 3B Spectrophotometric Determination of Protein Concentration+ X8 |+ R( p" u9 r! K( V* V8 {( P
3. 3C Dialysis and Concentration of Protein Solutions8 @3 X* M% V+ y+ Y; F) E0 ?/ @
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
2 i) [5 y1 u! r' u5 x- T5. 3E Silanizing Glassware
% ~5 ?% b: w7 c2 o6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
" D N: _3 A2 m4 b7 }7. 3G Micro RT-PCR
# m, _. c1 S, h1 `' l% F8. 3H The Colorimetric Detection and Quantitation of Total Protein
. H: [* S+ z; u. Y34. Appendix Suppliers
3 p3 {! E6 Q# h5 n4 `' @& I9 C1. Selected Suppliers of Reagents and Equipment% E6 w4 x' o1 W0 {
2 ]7 c- ]3 Z$ I+ n; b5 b1 {* V
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