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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑
6 m9 C. I$ P5 I. I
p: h7 _5 U8 i4 K# c3 g' M0 qCurrent Protocols in Cell Biology 2010年完整版 5483页
# z- v; [# A2 J/ t! `8 ]; B: Y$ ^4 t) a" e
Online ISBN: 9780471143031
* d+ t$ Q- Z$ }6 A! P6 [0 hDOI: 10.1002/0471143030% Z4 S- I4 ~. A$ ~+ q2 p3 o6 q
% n9 k# V& T" K2 I4 GTable of Contents
6 v) ]9 }. Z5 p7 }1. Preface# ]( c4 l! s* d8 a) K8 }) A
2. Foreword
; l1 \7 W# ^# n/ U: y& c3. Chapter 1 Cell Culture
# l+ ?- S! W! g1 B7 h, R7 @9 i1. Introduction$ p; x" j, G1 g/ j5 R6 B- |
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture0 s8 m6 Q: d1 b v# i4 O& r+ {- J
3. Unit 1.2 Media for Culture of Mammalian Cells6 M o; @ O5 w) \+ D$ f
4. Unit 1.3 Aseptic Technique for Cell Culture
; ^9 h* p. e3 K6 B5. Unit 1.4 Sterilization and Filtration
, L8 ~- f, I# Y6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
# V: g: P3 B( ~' h7. Unit 1.6 Media and Culture of Yeast
& A4 l7 y, c, x8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
8 X/ ?; }; u* |, e4. Chapter 2 Preparation and Isolation of Cells
' I- [7 J0 W0 c& b6 z- B# M/ B* e! L1. Introduction$ T! O" C3 k1 K( M% m
2. Unit 2.1 Establishment of Fibroblast Cultures
* Z0 S; B6 m% I: [. l3. Unit 2.2 Preparation and Culture of Human Lymphocytes
2 {3 v- s+ i$ T5 u5 u% C, D, t4. Unit 2.3 Preparation of Endothelial Cells
) j& m/ b; S+ x9 w2 P5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
# s3 C' p0 i4 d7 b0 j# ~/ j6. Unit 2.5 Laser Capture Microdissection
) ?2 \6 t- X/ |$ `7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures: C) ^4 K$ L7 }
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
1 E9 L$ _ g; u- ~) w2 A# x) y5. Chapter 3 Subcellular Fractionation and Isolation of Organelles, A5 a& M$ R; s* F; e
1. Introduction; D" U y+ V$ J/ N3 f
2. Introduction* I( V% c [1 U7 W
3. Unit 3.1 Overview of Cell Fractionation! |9 g& a% r" v F: ?9 f
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains- V, }# ?7 E8 j' P9 `
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation% x) v# y' i4 n
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
1 ^8 e( n# f/ W4 {7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
! g7 U/ {$ v! e, U8 N8 Z8 U. h% qCentrifugation
% U5 k2 H9 a% m$ V* x# U4 y+ A8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient$ p/ D: K* M+ y3 E4 \, U
Centrifugation
* p4 N# L) t; ?# f% N c: ?9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
/ V3 ?0 ]$ m/ v! B& [1 {10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
% J) F4 c- h3 [5 h# b11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient- R/ |/ M+ G; F# ?
Centrifugation6 y2 D- b3 u( Y, X4 r# i
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues" D! \3 D: K, I
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
5 p( L% J( N' ]4 T7 Y' B: A7 I14. Unit 3.12 Isolation of Synaptic Vesicles H2 X5 A6 p9 N0 V c; [) ]
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
" [+ w- e# Z( K# G- O% W16. Unit 3.14 Isolation of Melanosomes' Y O0 {) `9 y ^
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
. ?3 I+ N- ?" [0 V9 {18. Unit 3.16 Isolation of Mast Cell Granules
0 O) K$ b3 I2 U0 P9 S19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster- I# ^& V. _( l) p" D: D: \8 b
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
0 M( e7 u/ g1 p% X. m; u h21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
: s+ A l$ @$ r1 }* W22. Unit 3.20 Isolation of GLUT4 Storage Vesicles; b4 I4 \/ |, k$ T, Z; l
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
2 q4 l, K5 p( l0 p0 w' A24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
3 N6 m; m, s# y) G$ c. AFluids6 |( P" T. `' \" `+ ~
25. Unit 3.23 Isolation of Intermediate Filaments
$ B7 X" y! C: d3 ]& m6 g8 b; C9 Y26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
" A/ C9 s" ]9 Y$ W% n6 s6 E27. Unit 3.25 Isolation of Myelin! t2 v2 G5 y8 ^
28. Unit 3.26 Isolation of Renal Brush Borders
4 p2 B9 S# E- [/ `+ G6 O( D/ H29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
0 F) H! g n' i/ ` P, XFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
; y6 |# r, f! b. Q- Z& E. @30. Unit 3.28 Isolation of Amyloplasts6 U0 U0 k$ x# X4 ^" M0 a! ~
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins/ H8 m: k7 ^# G
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
& o* E9 P9 y2 B# @Centrifugation
8 Q+ W/ O5 K- z$ |% {5 P! C, V33. Unit 3.31 Isolation of Neuromelanin Granules
d+ q! g' r6 n# C& v! C. v34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
# Y }$ Y* D+ ^; }- t- W6 ^7 v& RDensity Gradient Centrifugation
7 z9 ?5 ?7 q, x. ?, d8 ^: |35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
7 r/ S+ k8 ^. c7 _6 ^4 ^; }36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation7 A* Y+ y' d1 l, [$ E/ f
37. Unit 3.35 Isolation of Platelet Granules
8 H7 W* a9 f7 |# W* \38. Unit 3.36 Isolation of Nucleoli
& O7 G9 b! S4 \* }5 o# m. B39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
0 q* y8 N$ H1 A# I40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
+ O, W: M) ]5 n# j7 `+ V41. Unit 3.39 Isolation of Chromaffin Granules; X1 a1 C! z( _5 n: u
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
8 D0 ^3 L0 K! ]0 q6. Chapter 4 Microscopy& ^/ ^4 q2 U, B
1. Introduction7 j( r: x3 B! ^/ O+ a5 H7 ^; W- T5 M
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
- `7 x. z6 W# Y+ d3. Unit 4.2 Fluorescence Microscopy
. \% L; E9 q8 x5 B* }4. Unit 4.3 Immunofluorescence Staining
$ ?. v! e# f- m5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria4 c: y. \ s4 V4 k: E& D8 I
6. Unit 4.5 Basic Confocal Microscopy1 h9 C" R5 [' T
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues0 r4 w" Y' L/ g( d3 N% P* l
8. Unit 4.7 Cryo-Immunogold Electron Microscopy0 U+ e) a$ i- {; {% E( ?5 h
9. Unit 4.8 Correlative Video Light/Electron Microscopy
& j9 ^, J9 V8 `! S5 y10. Unit 4.9 Polarization Microscopy
& j; M; {9 K4 J- T! b11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
! f# \" Z! G: s6 h/ C12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
7 N1 C" [1 _, `2 I7 e3 X13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
% m$ r$ V, p, \/ eEvents
; T" S1 w' a! P- H14. Unit 4.13 Fluorescent Labeling of Yeast/ s1 Y [2 j3 h" x. x
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy3 S; N0 f6 B2 T
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy0 b3 m( w* ^" g( m
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules$ g0 @( _5 u7 ~) y5 S& C( S
in Endocytosis- c0 ^0 p$ ]6 _+ F/ j, f% l6 i8 }
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
6 \8 I/ v) D. e. C4 I5 t19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development$ ^% W# b7 [. q6 D N
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
- l( Z# ~0 \, g: ^' {/ Z21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
8 a& v9 T3 h }22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
% R$ y9 J; I Z! B9 h( h3 |2 o' X23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
& j4 o% m; L/ W. z) c U24. Unit 4.23 Interference Reflection Microscopy8 s9 b+ P! A/ {! E
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
3 a; p3 W; L9 g* j* U6 C26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches: H& O' ~ c6 M9 Y! Y, S; h: T0 |
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
5 B5 ]( O- e. @& m- j% l7. Chapter 5 Characterization of Cellular Proteins
. g' m: ]0 ?1 e( P6 K3 d1. Introduction5 |- e# S8 d, w6 E' F$ m/ A. v
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
2 z6 F# k8 R o) e/ E% I% \, V j3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
0 t3 A4 z- V) C3 x) N% j! _$ y# x. E4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
; {* [) r. x0 H# f& ?3 h5. Unit 5.4 Analysis of the Association of Proteins with Membranes
! V& H1 L2 K5 e H/ |) k6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)1 y4 F0 O& S# u$ O/ w9 D( n" D
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
/ i) `- M) o6 E6 b. TSpectrometry
) @; l. r- n, G+ c4 w& W8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
& S& H0 d; m7 s% Q. w& I4 u2 dApplications
. _3 l4 C) t$ i% D* X4 u: q8. Chapter 6 Electrophoresis and Immunoblotting
* g4 \* z& O9 c* X, P, a1. Introduction
3 x$ e! [ l. C* h2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
5 D# q$ t& ~; _! M! \$ U3. Unit 6.2 Immunoblotting and Immunodetection
P# C8 a w( j' }0 j* I4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots* \: z9 s- Z! L# d5 f* k( U5 w& m
5. Unit 6.4 Two-Dimensional Gel Electrophoresis3 `1 I- Q4 V# u6 R! n& n
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
1 t& p4 ^0 v1 M! {2 r7. Unit 6.6 Staining Proteins in Gels
% O9 N: C( v& B9 e$ c' `8. Unit 6.7 Agarose Gel Electrophoresis of Proteins% Z# a2 \5 e" B5 G" S
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots9 r% \ W6 b5 Q7 e5 ?) p
10. Unit 6.9 Digital Electrophoresis Analysis! \2 Q& d( M! G7 P
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis @3 B! [# f! ] G' ^7 n2 p
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
: P8 Q8 J! t$ [, g8 c6 kSingle Cell Gel Electrophoresis (Comet Assay)7 Y1 R& m. k" {: H. h+ ]4 [
9. Chapter 7 Protein Labeling and Immunoprecipitation
2 z) x' ~, d% E1. Introduction
; P% o) ^- }- w+ B* ^2. Unit 7.1 Metabolic Labeling with Amino Acids2 ]4 D0 G4 w( g- Z# I* z& {
3. Unit 7.2 Immunoprecipitation% {4 ]1 A7 _8 A; T; X
4. Unit 7.3 Metabolic Labeling with Sulfate" a( s1 D; z* c5 g& c. T) @+ B
5. Unit 7.4 Metabolic Labeling with Fatty Acids
' d( X& \8 C9 y2 G) e6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
- W0 T. ^- N; M8 U# d8 w7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
" r9 T5 l" U: p J: K2 K8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
8 q( F4 Q7 A: Y' L& U9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
4 c4 s6 h/ P8 e+ r W; m' ]; }10. Unit 7.9 Analysis of Oxidative Modification of Proteins0 @, x" R) c" b7 s+ T! ?6 H9 L
11. Unit 7.10 Radioiodination of Cellular Proteins
; W3 t, ]: j2 }8 O! i2 T10. Chapter 8 Cell Cycle Analysis
h$ m+ d0 r+ X5 Q/ L1. Introduction8 T7 e! g9 P( E T8 \) H
2. Unit 8.1 Overview of the Cell Cycle$ x+ E9 R! |2 Q! f$ M# t
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
' _$ x# s, l [6 r2 Z j8 j4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
$ g+ d; t0 D! S1 z4 u5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
& ] a3 e0 U* l- _, l6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells6 O7 E! J' Q) ]+ |7 L
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry9 R" V& n3 k- g. ]
11. Chapter 9 Cell Adhesion
# m) W0 q1 K1 X E4 P; b" _1. Introduction- ^0 x, h4 F1 U6 E( a
2. Unit 9.1 Cell-Substrate Adhesion Assays
4 V, L% D1 _9 }8 S2 ?, m9 }* q3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
- k+ R5 h3 v; m9 x3 t' V( F# Z( ^4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
" N: [5 r5 Z8 m# i! d5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
# a' A! J4 ?6 f! y, j6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules: y/ v5 N6 W- e }2 ` {+ F# q. p
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions2 E# x6 h+ h" Q# E
12. Chapter 10 Extracellular Matrix
; l$ }& @) \5 [1. Introduction
9 {) g* _ D/ O/ \) F5 i- F2. Unit 10.1 Overview of Extracellular Matrix' _* A$ E1 L" f" ?) T
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors" R: B" ]% L* \/ B
4. Unit 10.3 Preparation of Gelled Substrates% t1 ~* K k" x
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR93 b, I; o `* V
Endodermal Cells* ?3 R9 x& {5 z$ p8 j! p0 q: n
6. Unit 10.5 Purification of Fibronectin. F1 i% S' z6 C1 D; O
7. Unit 10.6 Purification of Vitronectin
7 b* L" z: {: Y4 I+ E8. Unit 10.7 Proteoglycan Isolation and Analysis# O( L% C/ F1 U* m4 A! z
9. Unit 10.8 Matrix Metalloproteinases7 _0 c0 z" w, } |
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
2 U! {6 X' `$ x" p11. Unit 10.10 Purification and Analysis of Thrombospondin-17 D8 w: o) g) K
12. Unit 10.11 Purification of SPARC/Osteonectin
, U6 H: h- ^: q) J13. Unit 10.12 Analysis of Fibronectin Matrix Assembly7 O# `/ F8 C1 Z# V3 g5 n% T- J
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly- @1 Q2 j0 ]) l2 p. V6 Q
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
8 s- C7 g2 f7 l7 Q2 S, nXenografts" _7 S% C+ Q+ D2 V. Z2 G
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
/ U, A" @, P& D7 z& V0 G' X4 I' F17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties$ B, t3 l, L" J& h1 T
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
& S1 C/ a" U; a4 g1 d; L/ L5 UCell Migration
2 e, |1 j/ m- Y% ]. ~$ y, L19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix" D# I5 f7 {6 U3 T5 k: b
13. Chapter 11 In Vitro Reconstitution/ i3 V* ?" ?! }
1. Introduction
$ R3 \8 ]+ }0 _# @1 K2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
8 P/ b$ |1 l* z$ n, `) L3. Unit 11.2 In Vitro Translation
4 c8 S, b$ ^0 \+ F D7 G( t- |4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
: [/ i& N% A3 w+ [& `: w' Y# q1 \5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes/ P( l" u7 c7 u9 D, U% R" t
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication" ]3 S2 ?9 I& X4 N: l7 i
7. Unit 11.6 In Vitro Transcription3 O( i2 H, r; @4 C
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
2 m: d( u; f9 w7 c, [ B5 t# t2 J9. Unit 11.8 In Vitro Translation Using HeLa Extract
4 T/ D: K6 M$ r: _10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
) ?* Y0 C6 D7 Z+ A% k; [8 S11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
9 ]5 T6 R S% Y6 W( t12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
* R' D9 j" @8 H( }/ f13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts. j8 m9 E& P3 W& m
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
, |* E/ y4 r3 G7 d. d4 w: I( `15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes& ?. Y2 Y4 G5 ?, ^ i
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
/ P. \) r( o7 w3 [17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
+ t9 Q- k/ b& E18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts! i8 h. h! M1 [! P, q' t
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells5 Y" O: U- }( {4 g ~ ?
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import, I8 F! j( O ^+ z! w% ~! c
14. Chapter 12 Cell Motility" O t1 }* y+ ^% f o0 N$ x, f
1. Introduction
6 K4 b5 e) T% F6 m; W* P5 k* p6 r. Y8 m2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells! A$ Q, e+ B( b, n+ P8 T+ |
3. Unit 12.2 Invasion Assays
( u3 C- _9 J4 F4. Unit 12.3 Cell Traction1 t4 t- V3 }8 m& V
5. Unit 12.4 Cell Wound Assays; @8 Y H0 k. w7 q, r ?9 T
6. Unit 12.5 Dictyostelium Cell Dynamics
- A, z- g5 X7 y7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
4 s7 N( B* H" s5 i' Q/ ]* Z) d; x8. Unit 12.7 Actin-Based Motility Assay
) d4 R0 A8 ~7 h9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP! q( K0 g" q) c, s+ F( b
15. Chapter 13 Organelle Motility0 ?* W( |' K, D% b$ T- X
1. Introduction
3 W' j1 |% u9 @, J2. Unit 13.1 Microtubule/Organelle Motility Assays
2 }7 n a, k# ~! @3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin. f- z% U, g1 v* ?$ Q2 N; n
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP. P* `( T% s5 O/ k
5. Unit 13.4 Movement of Nuclei3 r) `, K% f' E8 R9 D8 B
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching$ E% g; A, ~2 r9 y# e# @- S$ d
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly/ Z! w( S" o0 S: p) ~
16. Chapter 14 Signal Transduction: Protein Phosphorylation% ~" K3 W5 w: j! N
1. Introduction
$ w7 |, E* e/ t) V$ ]2. Unit 14.1 Overview of Protein Phosphorylation
. a& B; A' w& t6 g" }: e3. Unit 14.2 Immunological Detection of Phosphorylation
3 U& r/ R* g# D/ w t/ ]4. Unit 14.3 The Detection of MAPK Signaling. ?# ^& l8 I9 w; L$ f7 j( C
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation$ ^ H6 e" V- g! t% j9 d0 v/ S
6. Unit 14.5 Phosphoamino Acid Analysis6 q8 v% C0 y w, Q3 ~' {
7. Unit 14.6 Determination of Akt/PKB Signaling, Y: A8 {2 L; L- ]4 q
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events" e2 G: q- T8 r& {& [0 r
9. Unit 14.8 Rho GTPase Activation Assays
3 {6 T t: H0 k9 _10. Unit 14.9 In Vitro GEF and GAP Assays
; v N4 k& Y8 k% Y" U, e11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
9 T2 [5 z& X7 m; x2 M& J& F' T# yEnergy Transfer (FRET)
# v+ i% P1 U4 |12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
& t- L3 |- R6 Q0 V# v; H" I13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells9 b2 p& ^. M9 l: r6 @! ^
17. Chapter 15 Protein Trafficking
+ ]/ F- u& i& V8 u$ @+ |1. Introduction8 G! _/ e2 {3 _! s
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways7 L8 z$ S5 `* y ]5 Q) D
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
5 T J, a3 u+ c! r4. Unit 15.3 Endocytosis: Biochemical Analyses: c# ]& y; B9 }3 |: P( Y5 [
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane& ]) w# u, f+ w# g9 g @
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells- @) E* F0 z8 F" Q
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum/ B: x& z* ~/ K# \; J. y/ w
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
. O9 S& m' H" ?9. Unit 15.8 Analysis of Protein Transport to Lysosomes
* \/ y5 ?/ H. Y; a+ k; a10. Unit 15.9 Studies of the Ubiquitin Proteasome System
) h e0 G! D, L4 J% j11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
: u9 j- C' C6 `+ J* x! @12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
$ p/ v" c: G8 j, L& ^13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells3 o& }$ ~) s o2 f
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
- r0 i' n- ^! Q15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
5 t7 R- d% ?" hEGF Receptor. R+ [' I+ R0 {* \1 O$ T% z
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
5 C! [& S' @5 S18. Chapter 16 Antibodies as Cell Biological Tools( S1 @! l: L" |* o: k
1. Introduction
! T/ A+ @: L- x0 ~( D6 _& t# r2. Unit 16.1 Production of Monoclonal Antibodies
' v' S4 ~4 _, O8 G" T/ m7 y0 I3. Unit 16.2 Production of Polyclonal Antisera4 G( m2 e+ m, d
4. Unit 16.3 Purification of Immunoglobulin G1 D# u8 l4 m; @" u; O/ s
5. Unit 16.4 Fragmentation of Immunoglobulin G0 ?/ \( C R) w- T) D
6. Unit 16.5 Antibody Conjugates for Cell Biology
' H& V% g8 Q2 p. i8 u: ~& |7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides/ x; L/ C$ \8 D- S' B& p& v
19. Chapter 17 Macromolecular Interactions in Cells3 v2 y' X& q; c8 p% C
1. Introduction/ K6 P! q5 B& ?( L. k( c7 y- w
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
2 k& C# Z- J5 {, {" I' kMicroscopy
( I7 s' q2 U: w* ~7 u( B3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
- Z' k+ D2 |* x5 x/ J- j' D3 Y9 \4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
5 D. v6 I0 U: R2 @2 w! p: m% b2 G5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries; Y0 C6 ~" M% @
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry* ~# q0 e$ A8 _: [
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors- J6 e- a- T" ?* R# w( R9 T4 g
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
1 I0 w7 o6 {7 O: F/ r5 p% u" R& l% uGenomic Sequences In Vivo
6 J) O o. k% [1 c; v6 I9. Unit 17.8 Isothermal Titration Calorimetry. X% @! f" B1 l/ k$ } O
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
: R: g$ s4 u/ I; h11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking2 [, h2 l. H! w
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
' t: f+ L8 F: ?Interactions (RFAP) in Live Bacterial Cells
# D. \$ ]. g6 b' _1 R/ F5 Q4 a7 c20. Chapter 18 Cellular Aging and Death
( M: F7 \) N9 O/ V m0 h- r0 I9 z1. Introduction5 m6 V- \1 K* h a* Z
2. Unit 18.1 Current Concepts in Cell Death9 e- D9 V- c& J" H; i
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
3 o6 F: ~% Z2 k9 H4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria1 l4 u$ `/ I6 l3 e; U% p6 u9 M j
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)! M8 w" i: d0 o, w9 ?& V
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death8 z7 x6 ~" `# ~' v i# y3 r
7. Unit 18.6 Analysis of Telomeres and Telomerase1 F1 a+ f/ B# |, m
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of1 r3 p2 O3 w9 b2 q. A
Poly(ADP-Ribose) Polymerase
9 ]; O' m' V- M7 ?6 V9 p9 M9. Unit 18.8 Flow Cytometry of Apoptosis" D# z6 }! L; C3 O1 Q4 ~ X# Z) c
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase s4 [$ u, a0 m) m. u4 ^; R# Y
Assay. q6 H! ~8 o' u% ?4 Y" R( P# ?& u
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
Y$ D# z* w3 u% `2 v3 O9 h9 k21. Chapter 19 Whole Organism and Tissue Analysis
1 e7 k ]/ u. `1. Introduction
0 Q' J/ m6 X; ], i# U2 o/ V/ X2. Unit 19.1 Overview of Metastasis Assays. `) a) O1 [, t) _8 H% Q
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis0 X6 R; n+ z1 ~2 ]) B
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
* [5 M: }- m& t0 P4 T! G! t: NExpression After High-Fidelity T7-Based RNA Amplification$ B/ i! Q; C0 N3 _4 z
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
) X, `/ t/ a9 ^/ M+ u8 X1 t6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model+ w* N# z, \- d: z: ?* l
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
6 G9 w, P/ q. W% Z0 q8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
- k; | j5 a% q4 ?9. Unit 19.8 Embryonic Organ Culture
8 O0 ^; W- g( U" [) v( f2 i- W% j9 C10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin1 Z( V: X# u X8 A
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice* i$ v& M1 d5 m( i
12. Unit 19.11 Generation of Transgenic Mice6 m* V: E- M2 X: F
13. Unit 19.12 Overview: Generation of Gene Knockout Mice# B5 s0 g- { c0 Q' D2 G: J/ v
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production* [' x0 Y+ ^8 C1 e$ _
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
+ S6 A4 M: H& w' f8 n22. Chapter 20 Expression and Introduction of Macromolecules into Cells# m0 x \. h! P4 [0 s- t9 O
1. Introduction% A8 }- {+ j5 S( K% Z
2. Unit 20.1 Direct Introduction of Molecules into Cells
$ o% M! K4 Z a5 ^$ N" G# K7 X3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System# L- f2 z. _6 x% d
4. Unit 20.3 Calcium Phosphate Transfection( T5 R7 P" {6 |6 }$ q
5. Unit 20.4 Transfection Using DEAE-Dextran
' I ?. y0 W+ D, s8 ?6. Unit 20.5 Transfection by Electroporation
4 t6 ~' A$ G* Q P/ [0 E2 L7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents1 ]! Y# l, ]% {# N$ p
8. Unit 20.7 Optimization of Transfection
1 y0 s% F8 Y' t) L9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System; g$ i7 p" k. G4 \! E) H% }# u
23. Chapter 21 Fluorescent Protein Technology
F- t; S8 x1 i$ H _5 A1. Introduction2 T; C( _9 N+ F( j4 T+ C
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
2 S; J: M* A) h8 A1 t0 s3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
3 p6 |8 H( }3 Z, O4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence! X( Q- @; S$ S+ M8 @) [' e; h' i) F
Complementation (BiFC) Analysis
9 F0 y/ W4 ?: O7 i. G& F- O5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology" x2 O8 J i. v1 e5 u
6. Unit 21.5 The Fluorescent Protein Color Palette
8 t% c# F9 ~& S) T, `% C! V7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins" `+ p d( ~. G/ P, d6 {
24. Chapter 22 Cell Biology of Chromosomes and Nuclei# a7 ? [! V3 F5 Z( @7 s
1. Introduction
* h7 Q/ J: F+ `" ?2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis7 o0 U9 P( T! [9 [/ a) ~ l I. s
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
" u+ _, K' o9 U. H4 n' A& i4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis3 J6 _ b/ C2 N$ L. U
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)" c* ?/ }) }5 p5 Z) i7 J
6. Unit 22.5 Multi-Color FISH Techniques
9 u/ R5 {6 `; C% e8 ~/ N7. Unit 22.6 Comparative Genomic Hybridization
' c0 m- h4 E+ W# ]; _8. Unit 22.7 Sister Chromatid Exchange V% B& F' L1 K+ j
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery4 j$ m; K0 Q. K& j) y3 V
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes) C5 S) N r- F! o5 u. s7 M: y
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs4 k9 Q3 |* X1 _
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
& M3 A, ^ s: q! q$ y13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells/ ^2 v6 s! g- I. {
14. Unit 22.13 Monitoring mRNA Export+ R; }: ~ w4 W+ W. ]
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-+ | }8 W4 `" |5 t- W' ^- A
Field Gel Electrophoresis
: v% Q7 W3 F T& I" }25. Chapter 23 Stem Cells/ Z* G" P' ~6 i5 \: f
1. Introduction
2 \, D" Q" g' V2. Unit 23.1 Stem Cells: An Overview' I! O5 J3 Q" k7 U0 j, h. H
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture+ H. k* [; a; }6 ^* F$ L
and Differentiation as Embryoid Bodies
: K e& L2 R7 X) ?$ q4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood9 t) ~4 i% L$ a
Vessels
$ e4 j" J1 ^5 |$ i5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into& U; s* f6 b$ H6 J; ~$ F2 Y. `7 k
Adipocytes1 A6 N1 c6 I2 q5 |: b/ E3 E
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation& Q* s2 H- f$ D1 o
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells1 A- |: B! E" W8 n8 e4 I, j7 }
8. Unit 23.7 Neural Differentiation of Human ES Cells0 H( x" i( C' E; D4 X) i" D) o. j
26. Chapter 24 Lipids5 E3 q: W% P T. E
1. Introduction( R# b5 L6 R9 x: W/ d
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
! C# _% E* n. o( t$ s9 ^- C/ d3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
, U; a1 c/ D2 T# v6 u0 s4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
: S% R" A8 C# g: ]5 R3 H+ w c7 M5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
! [* M* [( F$ } g. f27. Chapter 25 Nanotechnology$ A/ A3 ^& M5 T. w
1. Introduction) n- d7 w3 T' C9 h5 [! e& M
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes3 V! _0 @; _3 e9 \; x3 Z/ z
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
! @. K' H( J V6 T0 U% c8 }+ E- E! w" }& r28. Chapter 26 Viruses# h; G8 \6 t! ~
1. Introduction& g; R5 F1 f ~/ R) N' t5 x: z
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
9 v/ a" O& {7 ^1 k& L1 g3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
r+ y) E3 K) iCell Entry+ O% H& @" x: r4 K. {1 N) w ~! R
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection/ c* i3 C: O# V/ y4 \* x# b v( |
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
* |( o* J* n) X7 \9 u3 ~8 J6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission3 l; j1 \2 h8 W! W/ z5 w8 C- K' F
29. Chapter 26 Lipids
+ H0 x% w$ u3 Q9 j$ M( A1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
j. g0 c2 ` T) S* L1 N6 A% qReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication- e( N2 t9 V; K: J
30. Chapter 27 RNA-Based Methods in Cell Biology
% @; u5 U/ B3 s! x8 A1 @; q4 P0 m1. Introduction
, J7 B2 b3 S$ _2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
7 c. V! J- h9 \' n) Y( H+ Q/ N3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible3 M- [7 V$ Q; A M
Rescue in Mammalian Cells
+ @3 Q S* z. j2 B$ ]" v31. Appendix 1 Useful Information and Data
$ P; d. i; u- I' U4 J, ^- i- V$ F1. 1A Useful Measurements and Data
+ _& M' D! j T5 A2. 1B Compendium of Drugs Commonly Used in Cell Biology Research: y! s: H; i7 N6 G
3. 1C Identification of Motifs in Protein Sequences
, H8 L; f; U6 |3 }" }8 u. Y" v" M4. 1D Safe Use of Radioisotopes3 b+ I& p/ j5 v2 r2 I% ^
5. 1E Absorption and Emission Maxima for Common Fluorophores& Z5 m9 D$ j. O, W) k9 j% ?
6. 1F Importing Biological Materials
' p2 `/ o) W0 F% z' D; Y+ s7. 1G Centrifuges and Rotors7 q6 V& i! @) F1 k2 k
8. 1H Internet Basics for Biologists
/ A. X" j7 a: i0 \32. Appendix 2 Laboratory Stock Solutions and Equipment4 s0 {4 T4 i! l9 R. A
1. 2A Common Stock Solutions, Buffers, and Media
$ E: c [+ r4 n" S& s% ]& V2. 2B Medium Formulations
, L' B3 P# x! s+ I3 t/ Q3. 2C Standard Laboratory Equipment
3 t1 |( E: N# S3 |! A5 Y% O& T33. Appendix 3 Commonly Used Techniques9 e9 _" G% O ?/ d" z
1. 3A Molecular Biology Techniques+ B2 p5 M+ Q) B, y
2. 3B Spectrophotometric Determination of Protein Concentration
( q5 E- I8 H, M) X3. 3C Dialysis and Concentration of Protein Solutions9 W n% Q4 I( M1 m4 O9 ]9 O
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy& h% r1 v! V1 K+ Y& e
5. 3E Silanizing Glassware" Y7 o7 u' W+ u f1 _& ?- E
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
( Y! \ n7 r5 p" \- U' R) f7. 3G Micro RT-PCR6 @& W% h/ A R5 e F8 I
8. 3H The Colorimetric Detection and Quantitation of Total Protein% n. A4 g) x. \0 H, |; a
34. Appendix Suppliers% T, h* t- {4 N- _7 Q& a
1. Selected Suppliers of Reagents and Equipment
2 M" _% V4 v- `( E( o3 c5 ~5 {, z5 O) R) j6 j4 P/ U% p
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