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6 f; [1 ]( p7 H# w4 ZCurrent Protocols in Cell Biology 2010年完整版 5483页& ?% [* M% \6 |. z
: `7 I8 c" V: [7 z/ I( IOnline ISBN: 9780471143031
7 }+ ?! l6 z: I$ w- w7 ODOI: 10.1002/0471143030
. L# h# G; C: z. N0 L' I; v$ P
Table of Contents1 j- F* q- w1 o p8 a# Z
1. Preface5 b. p) V+ g' C. J. y
2. Foreword1 C3 D. h$ I4 f7 e
3. Chapter 1 Cell Culture
- b1 Y" Z& c, T3 f( q. A3 x1. Introduction
6 q, r5 q( T. c2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
$ r4 `3 B0 t6 s3. Unit 1.2 Media for Culture of Mammalian Cells
9 R" W. ~1 c0 i0 _4. Unit 1.3 Aseptic Technique for Cell Culture
6 p7 F- s J% ?% {8 k! b5. Unit 1.4 Sterilization and Filtration5 a9 g9 Q3 I \+ D# ]
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures+ I8 k% E8 E; y+ k( c) x
7. Unit 1.6 Media and Culture of Yeast5 S8 H2 w8 R' Q+ J, d; ^ q) M* n
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
+ [ s' c; N# [' e7 B! |- q4. Chapter 2 Preparation and Isolation of Cells9 K' @+ e/ E5 j/ [- U
1. Introduction! t. d3 z9 t0 m" S/ N+ @
2. Unit 2.1 Establishment of Fibroblast Cultures! I7 x0 P4 e$ H3 Q
3. Unit 2.2 Preparation and Culture of Human Lymphocytes* |' _* C" f7 }# l
4. Unit 2.3 Preparation of Endothelial Cells( ]5 `+ E. A3 o+ r+ ~& O4 R+ j
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
0 k' T0 T2 s4 ?3 B x: b9 w6 `6. Unit 2.5 Laser Capture Microdissection
, Z9 b- R- b; B" B: Y7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures, q# \$ u- F! L. {5 V: F6 b
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
1 y) C. f6 D5 B* ?5 d) V( ^5. Chapter 3 Subcellular Fractionation and Isolation of Organelles: P+ R$ O( D+ }6 z; X
1. Introduction
% F- G1 d/ C2 p H2. Introduction4 {) y! ^, h7 p
3. Unit 3.1 Overview of Cell Fractionation' v5 C* n7 f; Z0 ~6 y
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains$ O% h3 |. I8 j( K8 _* I2 j1 N4 x
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
$ l: ?# g6 v, n- d; k4 H- O/ U& J6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
" P& s9 [9 W' P+ j3 Y4 ?% c# ^7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient1 R4 |7 o( T6 J& V
Centrifugation3 k( E5 k$ T' w) J$ F/ i
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
1 V: Z8 o, d3 h7 O& xCentrifugation3 t( x' T G: P, v
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae2 `$ l2 Z3 v, @
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae% N5 }9 I. J( b7 R( i! ?4 e
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
- X1 S; Y9 I6 Q, E8 J3 cCentrifugation; t( s$ I* U8 {1 c3 c
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues2 s0 S- X) g6 \
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes0 G7 V) W6 k, C
14. Unit 3.12 Isolation of Synaptic Vesicles
- @- W8 M) L( x Z15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation) x; D- I, B" R6 z0 l# @, J
16. Unit 3.14 Isolation of Melanosomes
, S5 F$ T- K3 T17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation% y; E3 f6 X. j. G {
18. Unit 3.16 Isolation of Mast Cell Granules1 O0 e7 C6 @; I+ f
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster# E j. p D9 w) W( O6 Y; X7 S
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
3 }% L% e4 z* ]% Z5 l# y& w# C21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons; t2 P8 i% _" }6 n& P
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles- Q7 m _ d7 u
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes6 o1 `. r. _5 n# R
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
; ~6 j; g+ y r+ ]7 U- XFluids. m1 L# h. q% R1 ?0 H4 W
25. Unit 3.23 Isolation of Intermediate Filaments
- b/ {, j4 x! J0 ?! j; y% Z4 ^! O26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle+ j+ W( ^: q1 H, o0 n. |( `5 W
27. Unit 3.25 Isolation of Myelin
3 m/ `) y. ~8 I28. Unit 3.26 Isolation of Renal Brush Borders$ |( N5 I8 ]5 Y; b+ B
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
' C5 J9 G$ N5 G% rFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts5 X; z; F. T# [) A y- A$ F
30. Unit 3.28 Isolation of Amyloplasts; @' a4 U4 ~$ q+ E& l8 r" D
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
* s; f) r" Z! M0 o C! j32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic5 G2 ^0 I* V9 c
Centrifugation
2 O* z: ?) B) \7 O' Z7 m33. Unit 3.31 Isolation of Neuromelanin Granules2 }; x+ W3 ?+ _% \5 o$ |( p
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
; x. L( K) l& z; F BDensity Gradient Centrifugation
; L) `* N R T35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
# T1 [' B+ U, E1 \36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
. q. j, D" k$ @) f# W( t37. Unit 3.35 Isolation of Platelet Granules& R8 L$ i! Z2 I6 O- n4 K9 H. F- ?4 j0 q
38. Unit 3.36 Isolation of Nucleoli
; Y! @- j- j' Q( c0 b39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins- ]* G) R# F' Y
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates- S& z0 y* y1 X
41. Unit 3.39 Isolation of Chromaffin Granules
8 ?) p. i+ {6 w4 q9 a, s42. Unit 3.40 Purification of Ribosomes from Human Cell Lines2 z a1 V7 K' U, m% r
6. Chapter 4 Microscopy
: n/ O2 v; c: o* X3 c1 }7 |1. Introduction5 B2 r5 v; D2 X
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
8 v( ]. T2 B4 A8 [" ^ Q% V) ?0 X3. Unit 4.2 Fluorescence Microscopy7 V! u& J. K: J( [
4. Unit 4.3 Immunofluorescence Staining* I# B7 Y+ v" a! d7 W% n1 M
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria: J: A9 u+ d7 B. M2 p' h
6. Unit 4.5 Basic Confocal Microscopy
f0 c3 x7 I7 c0 O7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues1 @8 q8 K$ V# Y
8. Unit 4.7 Cryo-Immunogold Electron Microscopy
8 }) x3 I, P% u2 P b9. Unit 4.8 Correlative Video Light/Electron Microscopy
/ t, U4 ~' r/ d9 l, c10. Unit 4.9 Polarization Microscopy
' f& F' A6 e5 W11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
' B# P D- D$ z- D: u12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
+ ^ K; U/ g/ L13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
8 q0 q; R' w5 q! c, ]Events# S+ p) q: \' j1 I: {7 Y' o7 g: M
14. Unit 4.13 Fluorescent Labeling of Yeast
+ D! v0 {5 z# [! h) y2 q& k% ]. [15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
2 N; x# {2 J8 q+ A16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy6 X" h- R2 I8 I
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules, q8 X+ h# V8 T5 h8 t
in Endocytosis
f, G9 r% k" f6 L" C18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
$ t8 y0 r/ L% z, ^- l. m19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development( U; C+ x# H1 q' z
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
. j K/ Q4 T2 v" ]7 L21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions# s$ a' N& o& ] R* N' ]
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
: x" [; \2 L9 J) w" Y& H, b23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
8 G2 j6 U! [; ^1 z: e2 L24. Unit 4.23 Interference Reflection Microscopy4 v! g5 d: L2 Z+ l% J" W
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
3 ^# i% C$ W( R5 F2 C- O26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
9 A9 ~6 I% H7 \% K+ `27. Unit 4A Organelle Atlas: Appendix to Chapter 4
* ]9 E2 V0 ~( V: c4 e3 R9 J/ X7. Chapter 5 Characterization of Cellular Proteins7 x% p, L9 P; ?3 [3 T4 x! D {7 j
1. Introduction4 p( \1 }4 j: {, ~- j
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
& R* K, g0 b7 L) R7 H5 w* |3. Unit 5.2 Determining the Topology of an Integral Membrane Protein- j( w$ l4 k+ }9 k3 @% }
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
* o- W! E9 n: f! M+ W# D, D& X5. Unit 5.4 Analysis of the Association of Proteins with Membranes
8 X0 o3 u5 i5 D6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)4 u/ ]3 _( f& S R5 p. m' l
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
' l- c& A6 }- ]6 F1 {( X$ uSpectrometry
5 @' P, u: F' L5 p5 a8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening, p$ X& A/ A: c& n
Applications. ]0 i! w/ [8 k' V6 w
8. Chapter 6 Electrophoresis and Immunoblotting
4 L# W$ j6 Z6 _( j; n4 A# C* V1 ^1. Introduction" D" _6 J5 A! C! p" h; r
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins8 }9 J% U' |- ^) z* K0 H! M d
3. Unit 6.2 Immunoblotting and Immunodetection
! B2 R# N8 G8 [3 X, h1 C4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
/ r0 {; F4 ~5 ] b G3 W5. Unit 6.4 Two-Dimensional Gel Electrophoresis0 h5 G4 f( @+ b Y+ \4 S' z
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
, k; c' t! n; j6 i7. Unit 6.6 Staining Proteins in Gels8 x) _1 Y! t( q9 [/ Y; j- n% Z5 a9 A
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
9 z& D8 T) b! {, k9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots7 b) B5 k- A c$ l0 q1 L: i; }/ W
10. Unit 6.9 Digital Electrophoresis Analysis% B0 W6 y9 z) R2 m& R: F3 ]& H
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis0 ?' U8 z A* `- j
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
' n+ d3 F; o8 f- s' qSingle Cell Gel Electrophoresis (Comet Assay)
/ o, a n6 ?9 H" x7 D- O" x9. Chapter 7 Protein Labeling and Immunoprecipitation( L5 {. _2 p7 }; `
1. Introduction6 s6 [! ]# Y. @' B. b" n
2. Unit 7.1 Metabolic Labeling with Amino Acids
0 l8 q! o! K" T+ I" d+ @3. Unit 7.2 Immunoprecipitation: u2 H* \' ?0 k4 e9 G' s+ e! P
4. Unit 7.3 Metabolic Labeling with Sulfate
% J8 t+ Y6 F5 M0 j5. Unit 7.4 Metabolic Labeling with Fatty Acids
9 j! R5 _9 x4 c6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups( B6 }0 D- l* l% j, w
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins0 a, s4 ?5 k6 z' N. e, X
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
7 o/ y: R" }+ j9 \' u3 F9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars; N! U. F3 E3 h6 q6 l) P* i
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
- ^# n6 A, {$ d! O+ [11. Unit 7.10 Radioiodination of Cellular Proteins9 o* _9 `' K$ i1 w1 h) }- p) D7 C0 f
10. Chapter 8 Cell Cycle Analysis; _* R( B4 J. }7 ~, z5 H2 g
1. Introduction9 J' a m( z( W! T+ q9 P
2. Unit 8.1 Overview of the Cell Cycle; N, F4 P0 k4 c: t; k+ _
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
$ O$ d. O, B2 D+ z7 K1 L- D" K4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
0 w7 x4 }1 n, Q L5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry4 K! }0 G/ v1 r2 ~: X: E& ~! N% x
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells* T" n" m# t( i2 @+ n
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry- q; F! X9 N8 A9 R
11. Chapter 9 Cell Adhesion! T! n* W- Q; m8 C/ H7 V- o
1. Introduction
( M* h+ B' D2 w: I2. Unit 9.1 Cell-Substrate Adhesion Assays1 Q; i! W4 i5 m, b3 O
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
! r' {2 t5 z3 i; Y# a; Q' \4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion- U+ N& |: j' Z, K5 h: f2 I; g
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion8 e- S3 X) g: d6 S$ H- q* g; i9 s
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules7 D. [. Z W, w4 W8 L
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
4 A, Z- a% J6 H6 u+ ?- o! p. d1 `12. Chapter 10 Extracellular Matrix
/ B1 F! g# \- q% B; l4 u! j$ l1. Introduction2 h# z& X! m" G& g
2. Unit 10.1 Overview of Extracellular Matrix
. W& a1 a( w' I" ?3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
; t/ h( Y7 @) F( ?, |4. Unit 10.3 Preparation of Gelled Substrates
! ^* U/ S& J* Q n/ e/ m! Q5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR99 h4 C( e+ Q/ \
Endodermal Cells
1 R; V" x% y$ `5 i3 U( [6. Unit 10.5 Purification of Fibronectin
0 @& W1 I4 i& T9 [& u) N/ a! |' O7. Unit 10.6 Purification of Vitronectin( x% \8 |2 O2 K' l$ b: t
8. Unit 10.7 Proteoglycan Isolation and Analysis- R* W, d( j; Y) n8 R# G- u( U* L
9. Unit 10.8 Matrix Metalloproteinases
g+ e" }5 @: f* e( ?10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
7 Y+ z( W* X5 V; s e: F11. Unit 10.10 Purification and Analysis of Thrombospondin-1' m6 U- k$ h- B6 e! u4 p8 s
12. Unit 10.11 Purification of SPARC/Osteonectin
& I# \- C0 w0 u9 r: }, r) F13. Unit 10.12 Analysis of Fibronectin Matrix Assembly: D7 m7 W8 S, S& V
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
7 g0 U. _; M3 E' Q1 T3 j2 {/ o$ l15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
4 B$ ?0 g7 G5 a5 B2 @. H ~Xenografts
7 g2 s* l3 `( k, I5 }16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning% ]. i q g5 o/ r' @ I! U! t j2 i
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
- D7 S' W( J6 h! v4 P, ~18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
9 e# j5 g- X4 @% K0 P! MCell Migration" l# d. X/ R( S" m5 c9 D0 F
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix3 v3 N, j0 s$ _( N9 `- S
13. Chapter 11 In Vitro Reconstitution4 U" f* i: ?$ R4 I& a3 x
1. Introduction* l n2 R2 x4 U3 [+ h
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
' q3 @) L! p* b) y3. Unit 11.2 In Vitro Translation) V7 B5 _7 h- }. s V( j
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells. F' C' f5 [# Y E
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes6 S6 B# w' Y; p' y
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
" u9 S% J8 d% l6 \1 v7 F7. Unit 11.6 In Vitro Transcription
$ C5 c: k& F0 j6 `1 l( w8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells2 u! N# t8 y: f% C t! }
9. Unit 11.8 In Vitro Translation Using HeLa Extract
y; }7 L4 [9 H7 _% G9 d10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
5 }; b9 B* W* `" g6 W. z" \6 G3 w11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts/ h5 b5 r- M( s9 N
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
3 D( A0 H( D/ H" P* t+ l0 \& K13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts1 p. d5 i4 X8 ^9 @. `. [9 d$ x h) P4 W" G
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
# ~: g! M- s9 ]5 w15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
( d N1 T( q. B L8 n16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import' E8 b* ?4 j; h" w' b' w. N
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import& B! F+ V. g" v$ t
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
/ U# q8 T; {/ L' n: m1 z2 z: n19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells( _$ Z) M4 P1 W( _
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import: S! X; y& U* D- ]& P& w. y4 m
14. Chapter 12 Cell Motility4 ~# P- ^7 l: `/ `1 ~
1. Introduction
7 c z9 R- U- I( b1 T0 S8 G' e) s+ v2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells. ^% r5 Y1 }# {3 A$ w/ V
3. Unit 12.2 Invasion Assays
( I' B' {0 [5 Q4. Unit 12.3 Cell Traction1 x% f% K6 V: @8 R7 ^# ] |+ z
5. Unit 12.4 Cell Wound Assays
$ P% a+ S" M3 A* O6. Unit 12.5 Dictyostelium Cell Dynamics" v( u8 K' b4 S' Z; L- N
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
4 u T0 V" F. L& j! B' k- L* }8. Unit 12.7 Actin-Based Motility Assay5 G" u: L- E. r2 Y
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP) e( a8 G3 p, T, N
15. Chapter 13 Organelle Motility. e: X9 m2 S7 l2 H% G
1. Introduction
, j+ L$ R) m( {& l2. Unit 13.1 Microtubule/Organelle Motility Assays
4 y5 x# K+ f8 f, ]0 R l3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin+ I6 |/ R0 C8 j, V
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP2 I; o( A8 Z, F! }$ S
5. Unit 13.4 Movement of Nuclei- ^% u. p' z3 U( @3 F$ p* Q8 D
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching- d& o6 Z u1 R0 U! x4 V T
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
- h' ]4 c; H6 ^' H7 C. Y16. Chapter 14 Signal Transduction: Protein Phosphorylation7 F) [4 r) a# `3 }
1. Introduction" z) w3 u9 Y w2 Z# X! v, m
2. Unit 14.1 Overview of Protein Phosphorylation
$ ~4 o4 z! s: [* t0 f7 o6 ^3. Unit 14.2 Immunological Detection of Phosphorylation- s' M% `! F' A5 ~. M% f( p
4. Unit 14.3 The Detection of MAPK Signaling
. l/ A, W8 P0 g; E5 L: _# \5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation5 t' q" t' M% M( x( x
6. Unit 14.5 Phosphoamino Acid Analysis0 N3 Q; ]% Q0 J) l c
7. Unit 14.6 Determination of Akt/PKB Signaling2 V9 F+ u4 _3 A' K: K
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
) i8 V, d2 k" B+ s# \: s; ^9. Unit 14.8 Rho GTPase Activation Assays
3 Q# o8 `2 N5 X1 H, l6 p& x- y10. Unit 14.9 In Vitro GEF and GAP Assays5 _( I$ C! ?; q3 {
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance5 Q% W& B' b! {/ P/ H' u/ R) O
Energy Transfer (FRET)
0 ]9 w; C4 K7 d; q; r/ v; ?12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
. H, \- Y# Y1 o/ i' W" v" v. f13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells" }7 [3 A' Y' b! v8 g! r" @: q: f
17. Chapter 15 Protein Trafficking4 u8 G" ^5 b Q _* Y6 a9 R. |0 z+ D
1. Introduction% C* K8 E3 A3 X1 {: u' b7 d
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways. o* K0 M5 j' p6 B8 w
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking- L5 t; }& x" m
4. Unit 15.3 Endocytosis: Biochemical Analyses
. J( L9 O& B% s0 c5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
, F5 ?: g3 v9 B6 |4 V6 [ N8 v6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
6 r/ U! x# p8 v7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
& q# f3 @7 V9 e8 {4 h+ y& v* n8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
8 M% e- q" y3 O& {3 }7 H9. Unit 15.8 Analysis of Protein Transport to Lysosomes4 T/ f% g d5 l/ d! D/ \1 @
10. Unit 15.9 Studies of the Ubiquitin Proteasome System8 L/ }0 @6 K3 y1 H( H
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
0 f; a4 W0 h* V( V- Q12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
% W( x$ L8 z3 R13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells! \( t6 i8 K% h; U' t' t! s. f
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
G! h' w# M0 q$ [7 @& `15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and" `: U" D) s% {
EGF Receptor
7 E8 `3 M% l7 p2 e% K16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers9 y. B8 G5 V" E3 Z! c3 T5 h- T
18. Chapter 16 Antibodies as Cell Biological Tools. l$ S3 `2 q4 ^( B# s2 U+ F4 h
1. Introduction
: J6 p0 s0 ]3 U4 `& w1 m2. Unit 16.1 Production of Monoclonal Antibodies) ]1 g, c/ q7 ^0 v' I
3. Unit 16.2 Production of Polyclonal Antisera
6 c) N4 g. X4 F4. Unit 16.3 Purification of Immunoglobulin G. i# ~8 F+ E9 e8 |- v: z0 | r, v. [
5. Unit 16.4 Fragmentation of Immunoglobulin G
9 G( k6 p9 b, t' D( n# ^* j9 v6. Unit 16.5 Antibody Conjugates for Cell Biology
- z3 z' Z' X* W; _( p/ A7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides1 ]" e4 [; f d/ @0 B
19. Chapter 17 Macromolecular Interactions in Cells1 c! t) V; v9 a' Y, n
1. Introduction" ^ ?/ V; D8 q# C {- v2 r
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)/ Z" y1 y' F# Z; [: K
Microscopy5 \% S9 J& e0 z Y7 ], j' j' r
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
$ Z/ u: R: A' [9 h; t8 R4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
+ m( p- V0 h( F7 o" ^6 `1 `5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries. Z7 J, v% @, p/ v* g: e* B
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
6 f1 [/ [& ?) F" H7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
8 k5 C3 b Z2 E0 G( ~8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific( H% p1 @; |# ^$ D
Genomic Sequences In Vivo
2 a$ d+ Y# M4 X, I: t5 N/ R9. Unit 17.8 Isothermal Titration Calorimetry7 | F8 F! G8 v3 c2 p2 z
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
# X2 O( ?1 `3 y* W8 F11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
) N* `& M- w( p7 v12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
3 f4 q& l& S( q% J- yInteractions (RFAP) in Live Bacterial Cells$ D7 q/ V1 D0 M& n( ]' n
20. Chapter 18 Cellular Aging and Death
3 b: D- I1 A/ k) t; v0 n! v! b8 f1. Introduction7 S8 d1 f' E+ G4 A" A( m
2. Unit 18.1 Current Concepts in Cell Death5 O" n5 Q9 e. l( t
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
" h; g; j, I6 u% D4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
- T" z7 D3 Y1 h/ m( Y( U5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)& u3 r3 Y( m( V
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death3 c+ |5 W- p: _7 I6 K4 a: ?- |
7. Unit 18.6 Analysis of Telomeres and Telomerase
$ x7 W7 m# v5 i- y* U6 L8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of1 w, f9 Y1 s! d3 j3 w7 t
Poly(ADP-Ribose) Polymerase# Q0 }, n B* g# \- q$ s4 l
9. Unit 18.8 Flow Cytometry of Apoptosis' \. z0 a( s- ^6 H: J
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase7 |" }; e2 I+ Z2 D$ }
Assay
5 R# {; z9 x' N1 g11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
0 O; M; E" I! @0 ^! S. ?21. Chapter 19 Whole Organism and Tissue Analysis
- _) M8 n9 D' T) i1. Introduction
" g; x3 T! @" J, Q+ \ U' T2. Unit 19.1 Overview of Metastasis Assays( e% R5 i% k* N/ k3 O/ @. F
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
( X$ K( o) A& F, B4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene) \% u u3 Z. C9 W! ^0 c
Expression After High-Fidelity T7-Based RNA Amplification9 B8 q1 I% q+ W" r
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
) E6 {" I) E6 ]5 f3 M6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
3 @# Q4 V& g4 T7 F- L9 X c7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
2 k/ |( J4 b3 E, L* r2 e C8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
. `5 L. K. {) f$ A$ \/ x- K0 G' A9. Unit 19.8 Embryonic Organ Culture
4 }3 Q/ G# a1 h8 w10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
# @, [& x2 c" ^2 H: ?11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
+ B0 V- U7 x* h$ X( y+ v12. Unit 19.11 Generation of Transgenic Mice
- w! p& r" {) E3 n; t13. Unit 19.12 Overview: Generation of Gene Knockout Mice
! L; \/ ~- H2 h14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
; R! y9 Y+ S( I6 q4 X8 i15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
u- s$ E% `/ _+ ^ [( M22. Chapter 20 Expression and Introduction of Macromolecules into Cells
* @& j B7 g- s1. Introduction. l+ q0 n' [# A& P7 ^, D9 s+ i# v
2. Unit 20.1 Direct Introduction of Molecules into Cells
/ Y. G/ J: y+ Q1 [4 ]3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System5 m# n8 O7 R4 I% l5 I' _
4. Unit 20.3 Calcium Phosphate Transfection
9 I" r0 ?" e, g5. Unit 20.4 Transfection Using DEAE-Dextran
% r: U0 b' M( K& Y6. Unit 20.5 Transfection by Electroporation' J5 s3 g1 t7 @- G+ i; G
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
S' z, a$ z- }) o H$ e! X- v8. Unit 20.7 Optimization of Transfection2 @! V9 C, {* Z& f- n
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
% u# s; Z5 O2 d$ Y23. Chapter 21 Fluorescent Protein Technology
( v! O0 |; Z( o/ r& m4 I5 \1. Introduction
I# T b. ]( f- j2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells1 {3 K6 W6 w7 C" x
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
- ?% f# K" y7 O$ a- i1 [. j4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence9 o* `& V) i2 D3 G
Complementation (BiFC) Analysis! F; P$ y- A3 k& d
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
; v2 C4 R5 G3 U+ B7 `% f6. Unit 21.5 The Fluorescent Protein Color Palette
, c' \* g- z! D- L7 v/ W6 P7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
; Y% o6 [( U. U( z2 a24. Chapter 22 Cell Biology of Chromosomes and Nuclei
! `/ A* t2 t5 X' N C9 R" F1. Introduction
1 I, h: ?' ]3 J2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis% @2 I; s* u' ^5 _% [; }5 H8 A/ R
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
! F" Z' \2 _9 e$ i5 `4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis5 t" j! q% Z) @9 F8 J
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)0 g* Q3 {8 C' |9 H/ Q: c
6. Unit 22.5 Multi-Color FISH Techniques4 A/ s7 z( u- a8 M% c7 {
7. Unit 22.6 Comparative Genomic Hybridization
: v v; C$ C- u/ X+ N9 \" M" E8. Unit 22.7 Sister Chromatid Exchange U' g2 [: [* k
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
1 Y, t; a4 T1 [. L! H10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes6 S7 ^8 @. Y+ d; |9 _
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs$ d8 Q# u7 |; v- J6 \8 Q
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
! r: i+ {: w: f, g: Z$ e13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells F( o7 N0 Q- {2 N( n
14. Unit 22.13 Monitoring mRNA Export9 X' c, m' i/ Q
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
& `1 H- e/ c% A) n. JField Gel Electrophoresis# L& s: [$ B( f8 B8 }
25. Chapter 23 Stem Cells$ [/ D) Y' X5 q& i# _% c3 C6 l1 q
1. Introduction
. f7 _9 W Y" J2. Unit 23.1 Stem Cells: An Overview! J( ^- x' o$ l. t: C6 W
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
7 U' z: H+ L4 y( o0 gand Differentiation as Embryoid Bodies8 E$ {% t9 B% j( D# v+ P: Z9 B
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood F2 X( U0 k9 H# H( g
Vessels
$ {% ~, ~3 a m3 v& p. l$ S5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
( K* _, o1 E5 f; b: b& C1 {Adipocytes
# E" O5 |9 I& L/ S% y# V6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
( w# L/ H8 i/ U' S6 B7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells: ~7 D) z# i; Y0 o
8. Unit 23.7 Neural Differentiation of Human ES Cells$ b/ U+ m) C; e3 k2 }
26. Chapter 24 Lipids7 @" Q5 z" p; c `
1. Introduction
. W# J. F0 i& s% O4 o2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
/ y+ i% X" z7 \# L0 \3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
8 d. b1 B/ b4 }1 {4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
8 x) c7 K5 v4 J E1 a1 \/ F5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
- A- Y$ k: f2 d2 l27. Chapter 25 Nanotechnology
3 n* f% b! G/ D7 U2 E' y8 L1. Introduction
! g$ w- Y0 Z1 ~- e" ~. A/ {8 W* d" ?* W- a2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
( f3 j, z0 i. ?3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
: I' Q, L$ s7 P- {3 G9 Z. J28. Chapter 26 Viruses
2 N3 c+ O0 H0 S) \& n1. Introduction/ {1 D |/ J4 r) \& d
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
- ]1 @# f% f& I9 j3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of6 G [+ t: r3 c: s3 H- Y
Cell Entry: I! @ x3 t, u: x, K
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection+ `/ T- N8 s" P8 b. v! Q
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
5 K& g2 p) o. C0 o3 o6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
* _0 m( t4 M; [0 [* O0 `8 P29. Chapter 26 Lipids
; B$ Y4 ]6 W6 z1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by0 O+ X4 K8 @2 g2 X4 D4 _1 q, U
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication# q1 O" m$ Z. ~8 m
30. Chapter 27 RNA-Based Methods in Cell Biology
* L, U5 w3 X5 ^+ X7 U2 X0 }1. Introduction
4 [9 @4 a+ G" Q- I& ?, X. m1 o2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs P. r" k& [" _7 C! E% v' H; k
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible. ?9 b- j7 V4 P6 n
Rescue in Mammalian Cells) j0 n. N8 M1 ?( W3 Q
31. Appendix 1 Useful Information and Data
: K0 m* s2 T# H) j' w1. 1A Useful Measurements and Data9 B$ M; L+ h( p8 Z4 ^3 A8 X
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
2 D2 D* z# T2 T" a3. 1C Identification of Motifs in Protein Sequences
5 `4 Q% _2 T% q2 V' L. G; U4. 1D Safe Use of Radioisotopes
; Y- V8 g& ^4 y7 _5. 1E Absorption and Emission Maxima for Common Fluorophores
6 U' f% o! E/ u3 |6. 1F Importing Biological Materials1 ^5 ^: M# u& L# C* m8 X" A
7. 1G Centrifuges and Rotors6 D- u9 w( C( c% q: k3 x3 f" A
8. 1H Internet Basics for Biologists7 \1 Z5 J1 @3 H9 B0 `+ u4 e' j- k& _: X
32. Appendix 2 Laboratory Stock Solutions and Equipment
/ f/ a* h6 S8 c6 ~% ]1. 2A Common Stock Solutions, Buffers, and Media0 k- G2 I8 [; W9 {7 |* B/ C
2. 2B Medium Formulations, c; _6 J: M# ^4 I: { @
3. 2C Standard Laboratory Equipment* t2 [1 p6 _4 v( E9 L
33. Appendix 3 Commonly Used Techniques. }2 M' I0 N( m% ^0 L" P
1. 3A Molecular Biology Techniques
% y9 t% U: e8 i7 }2. 3B Spectrophotometric Determination of Protein Concentration
; A. i0 p8 h; [0 _* X+ e3. 3C Dialysis and Concentration of Protein Solutions
D; a2 L2 y1 f- m# P4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy! G7 N3 m1 J4 w6 V! r
5. 3E Silanizing Glassware( Z/ c! r5 \9 S' W% b
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization1 J' q3 w% [( Y7 i( H1 t' E
7. 3G Micro RT-PCR
0 z1 e }) P+ F. g4 o5 p* l5 F8 _8. 3H The Colorimetric Detection and Quantitation of Total Protein
) n9 L+ i# \% _9 \4 e3 e34. Appendix Suppliers
% Z2 L6 j' f; @0 {4 ]0 N/ J1. Selected Suppliers of Reagents and Equipment
' ?; e2 v' q4 y; S8 F+ Q. ?# |4 B0 I5 B
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发表于 2011-3-8 15:53
