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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 # {; b8 S# Y( J( Y: w7 \. q- h9 q
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Current Protocols in Cell Biology 2010年完整版 5483页/ o/ S+ q$ ~" ?* f
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Online ISBN: 97804711430318 D6 O* r. \ P: z
DOI: 10.1002/0471143030% `' w8 t# {; Y, r. b2 k; ~
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Table of Contents) e6 M* ]1 g! k; s" @2 q5 O
1. Preface
- z: m& u, l) G# _ N2. Foreword
* r r( R7 D, o3. Chapter 1 Cell Culture
$ O2 m+ K. _$ | I# V" S8 L1. Introduction
. |& R& u+ ?. k; l+ c) m) T$ w8 A2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture; t8 C0 P/ F- Q9 Q/ a" o7 J
3. Unit 1.2 Media for Culture of Mammalian Cells
7 w3 g8 V% G* v$ {6 D4. Unit 1.3 Aseptic Technique for Cell Culture( t2 e+ o9 k4 [1 q
5. Unit 1.4 Sterilization and Filtration
2 ]+ t' C; j8 m1 K7 s- B4 [6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures: q9 x- q; P! A; e5 T3 d4 h
7. Unit 1.6 Media and Culture of Yeast2 }1 ~- j5 y# d6 v+ G8 P
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
5 K8 U5 V4 _: \4. Chapter 2 Preparation and Isolation of Cells3 ], ^& v6 P3 P9 l6 b$ ^
1. Introduction
; G3 _5 ?* e6 n( c8 X! j# j2. Unit 2.1 Establishment of Fibroblast Cultures
9 M7 _% P4 S% O. u% N4 Y) \3. Unit 2.2 Preparation and Culture of Human Lymphocytes; @ w: ?& M. B5 i7 F$ h
4. Unit 2.3 Preparation of Endothelial Cells
2 i! z. G- J, A0 m5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
w( x5 b5 d3 ^0 ]/ i, n" L9 ]6. Unit 2.5 Laser Capture Microdissection
* T, a' O) N# _5 o) ?7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
4 ]0 ^0 r; _9 b5 D& r7 t" r' T4 h8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells
a% w0 Z5 {6 x( N; c1 g5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
# n, T9 K6 ]5 ^, L7 V1. Introduction
8 I! b$ G% h/ _' ?2. Introduction$ G/ j' h( m, ]4 S, S; D. d2 W
3. Unit 3.1 Overview of Cell Fractionation' q3 B2 z2 `* r) e S
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains8 K5 Q3 w% H4 r; ~4 q: e7 ~
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
6 W. f6 q5 |8 ]1 R1 j8 _6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
7 r' Q' k+ W: w- Y/ M/ I7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient
: K4 s1 u! \/ s/ k S0 V: a2 kCentrifugation6 c: s& n) [ r3 \
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
5 x: H" Q$ f. J6 s* ~) }8 e: eCentrifugation
5 c2 W2 y* L( ~5 G3 s) o9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
( h) |5 A0 [: l0 f u10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae" h1 {* T% w% c) P6 v& z. |, U
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
9 e6 V8 f. m1 g4 n4 tCentrifugation
' C# c, i1 g6 W- C% E8 q12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues$ y! p% r5 X R8 ]
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
* q* M. {+ ?9 b! l* E1 b9 d14. Unit 3.12 Isolation of Synaptic Vesicles
* w; J% A5 g% p! J9 c5 h5 F. \+ p15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
3 x% f* h2 L: v6 V16. Unit 3.14 Isolation of Melanosomes
" L: ^9 g" H7 @+ h3 ~) F17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
& |) x Z- t: V% i) T; H18. Unit 3.16 Isolation of Mast Cell Granules
4 |1 D1 F; B$ e% D) l( B# r; v19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
; x. o% ]. ^3 `$ R% @7 w) m5 W20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas Q9 q/ \: {! J3 ~
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
9 t4 C/ T: Y9 J2 i8 R22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
5 d, ~, _, b& v0 S. D23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes8 A5 O4 ?* ]3 P
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
4 w# r+ O! q* x. r9 W& w p2 K$ |Fluids$ y- m8 Y- X# N3 o7 i& q
25. Unit 3.23 Isolation of Intermediate Filaments
0 c9 Q' q! [* g+ G4 j. b7 z$ f26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle4 a8 ~ y5 V% I( R
27. Unit 3.25 Isolation of Myelin0 `. N7 {$ S1 I/ I5 Q% N c
28. Unit 3.26 Isolation of Renal Brush Borders$ k+ \8 j8 D" k0 u4 ?+ {3 |# d
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
- W L0 ]+ ~" v$ n( a! Y1 y8 rFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts* D2 \4 N1 m' ?1 X; k. E$ I
30. Unit 3.28 Isolation of Amyloplasts
! t/ j4 C4 k& E% g31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins8 n6 ~$ V$ Y2 {% S5 r3 o$ {
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
{/ V9 X/ G1 b8 d/ ]; ?! V, HCentrifugation @! _3 E- `- L' ]
33. Unit 3.31 Isolation of Neuromelanin Granules/ |1 G5 P, L8 X: v& r
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and3 q( O d3 G4 c" C! s
Density Gradient Centrifugation$ t1 s# M$ i j! f3 y6 H7 ` C
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments8 p0 }/ f- @" e) `# u
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
0 a& H3 m6 R/ P- u ]3 a37. Unit 3.35 Isolation of Platelet Granules
* h3 u; Z; {# e9 t# p38. Unit 3.36 Isolation of Nucleoli+ y v- U7 z. o: d3 Q. H
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
' e- ?4 X% w6 f4 w2 k/ d40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
; d' @. Y- k) R41. Unit 3.39 Isolation of Chromaffin Granules
' x( b% B a H2 _42. Unit 3.40 Purification of Ribosomes from Human Cell Lines4 O- A3 x2 |/ y y* @) R5 n- g, ]- P
6. Chapter 4 Microscopy
6 W* |+ w# M. e3 H4 k1 V/ w' a$ A1. Introduction' q: w, B1 `; `; j' {% [
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope8 T: V' ?/ [9 h0 T2 U) ?
3. Unit 4.2 Fluorescence Microscopy
: E3 K4 W* Q* x+ F% r. l0 I4. Unit 4.3 Immunofluorescence Staining" g/ A* n) Z0 u' @4 I; V& |' L
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
+ ?- y! ?6 M& A6. Unit 4.5 Basic Confocal Microscopy- M8 A3 P# w/ S9 \; j
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
4 d7 N3 W5 O! H9 N8. Unit 4.7 Cryo-Immunogold Electron Microscopy# z1 ]# p) h- r7 \; x8 [+ C7 t' p, ]
9. Unit 4.8 Correlative Video Light/Electron Microscopy
- l7 [1 c4 H- ?1 r$ F! F# f. u2 D, k' s3 b10. Unit 4.9 Polarization Microscopy
& p' V7 g- `% V4 |1 ^" r11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells& F6 u8 P" ^! |5 t5 G# s6 x% _
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues0 ]: R1 E& o, f" Q
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface, r) T: L+ B u2 W1 T% ?$ k
Events. @) w. g$ S$ i6 k
14. Unit 4.13 Fluorescent Labeling of Yeast
! p6 y( l7 O; Z# t5 l15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy$ c/ _. O: Z" Z \
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy$ \5 \( I) J t+ H6 Q5 u
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules/ n- i7 U$ K; g
in Endocytosis
, a* e3 L+ }1 e1 Z- s0 K# g18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
9 b. y! P! Z( S6 l# O" s6 T' g19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development- U7 ~2 U5 k/ X3 [: c0 u/ f
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
6 z9 l0 A; ?5 C6 ]# G( {21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions C7 r* I- i' i8 |" _: a" i
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
: \; E& W& G( J1 c$ R- d5 K23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics+ J3 q8 r7 h" ^+ H$ {
24. Unit 4.23 Interference Reflection Microscopy
( S0 ]& v$ Y0 q* G6 |25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
_) f, k' B; o4 X9 S; o26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches4 u$ D4 f+ R% I5 }1 Z9 \) ~) U3 q
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
, ^1 f, `# s% L3 p# W7. Chapter 5 Characterization of Cellular Proteins
1 \8 u/ j) s! g% R# |9 o6 i1. Introduction) k* R( V8 l; U4 x
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
* B, M" Q# d8 C7 k% _$ i1 U- w" e3. Unit 5.2 Determining the Topology of an Integral Membrane Protein; Z& |% |5 B- @+ P3 I
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
' o) {1 F7 R$ r/ J: I1 Y) H5. Unit 5.4 Analysis of the Association of Proteins with Membranes
! \( T9 N0 Q' T" t) K _+ h6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration). w! r) m; ?7 w7 w3 O7 ~) E
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
" K6 I! Z& P2 {* h1 f/ x! X! ySpectrometry' v) W4 F4 Z; i, r
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
: T7 P- e: H" k; e5 r) Q. l8 N7 XApplications
" m' F0 r3 \0 i8. Chapter 6 Electrophoresis and Immunoblotting
' ?5 i& M, J% j+ {! C \" C1. Introduction4 C x( g |1 i" w3 e
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
9 U5 y$ k4 C+ h* h3. Unit 6.2 Immunoblotting and Immunodetection+ S6 I/ P9 I2 L, j4 C# w$ R: l
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots& R. G: p6 \. R" e* E5 s
5. Unit 6.4 Two-Dimensional Gel Electrophoresis: ?. Q9 E$ z1 S7 @) j$ n( {& f# {
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
7 L9 C; b" ~$ g5 H* s7. Unit 6.6 Staining Proteins in Gels
P9 r- X8 a# A7 K+ k8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
; `9 h7 b; j- K% K0 r) g1 d9 q9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots$ `4 y7 ]; p, B( v B) y
10. Unit 6.9 Digital Electrophoresis Analysis2 a! O. c7 C0 o# p7 \$ p
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis2 u& ]/ ]3 c) m5 ]0 r
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of( z# a* e) v" B+ h3 c
Single Cell Gel Electrophoresis (Comet Assay)
+ F- S3 E. Z% V# P9 w9 B7 A! Y# b9. Chapter 7 Protein Labeling and Immunoprecipitation9 D# k8 H% R4 d Q" m$ h6 n
1. Introduction
5 d" ?. m0 F& D1 u0 c& y2. Unit 7.1 Metabolic Labeling with Amino Acids
, z! f0 p- O6 T1 O* k3. Unit 7.2 Immunoprecipitation# G5 |* f% ^/ l$ l& _& B! W/ _
4. Unit 7.3 Metabolic Labeling with Sulfate
: l- ~5 W+ N6 W5. Unit 7.4 Metabolic Labeling with Fatty Acids; V! }9 Q. J! ?6 c Z i
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups5 ? c0 E7 ^" O% K k! N8 [: M
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
2 H# |2 n- [4 _! d; ]8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
* k8 P9 ?8 N7 v& R9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars, x$ A1 z4 g, {6 s. K
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
. S; W: d9 Q; Y+ M, _ V1 y11. Unit 7.10 Radioiodination of Cellular Proteins" O$ O# N0 v, {8 p" g& P
10. Chapter 8 Cell Cycle Analysis
6 z) R/ {: w' p& Q0 Q* Y3 O! e% C4 F1. Introduction
- h7 A, g% p1 d( u: L$ B8 R2. Unit 8.1 Overview of the Cell Cycle
& _9 S, j: J/ W) t! @! h7 {" b3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
2 P6 n' y; B; O) D# K& l4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
, |9 j+ S+ d' d5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry, B3 U. _# t4 y2 A$ N2 `$ E8 c/ W5 @
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
3 x9 k3 o9 I7 C7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
$ |! U* t/ e( N, D; Y11. Chapter 9 Cell Adhesion
0 _; J) ~# e4 Y J( O; { `1. Introduction0 ^4 E+ d6 n5 N1 o6 s0 W' C
2. Unit 9.1 Cell-Substrate Adhesion Assays: J& C% R& g1 c$ [$ x/ [2 |2 r7 k
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
% ~5 [2 s7 f- y$ p% \# W4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
. n3 a0 }+ @( l m# |& L+ B5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
- F, p7 e- k8 S' J/ |6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules: Z9 _/ B1 I+ K9 X% o6 x9 O
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
8 y$ ]- K0 m) R6 `9 C12. Chapter 10 Extracellular Matrix
, L% F7 R4 A O Q/ ^9 t6 E7 N1. Introduction# n' p2 K- ]$ J! p
2. Unit 10.1 Overview of Extracellular Matrix, Q2 ~3 h7 s, I$ s
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors* s: U# u9 V8 C+ t
4. Unit 10.3 Preparation of Gelled Substrates
% r. n( w/ e* N5 U# U/ m5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR92 s. h; N4 m$ u# p0 q0 Z
Endodermal Cells
# J% T4 [$ _, J! Y6. Unit 10.5 Purification of Fibronectin `: l% [% j. a0 g7 |# N
7. Unit 10.6 Purification of Vitronectin1 s' q" Y7 b7 i: D5 d
8. Unit 10.7 Proteoglycan Isolation and Analysis
0 ^5 W6 r [6 \0 e- I9. Unit 10.8 Matrix Metalloproteinases' g2 |# ]; L- b2 S* }
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
* l4 A: b* r0 m7 }& F11. Unit 10.10 Purification and Analysis of Thrombospondin-14 \4 W4 K4 |8 {5 I; {& _
12. Unit 10.11 Purification of SPARC/Osteonectin
# c- g7 E( y& A* b13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
8 [: p4 \# d/ ?& h2 y6 v14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
3 P4 ~% \! K0 ^1 Q8 { S8 P15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor
2 U7 Y0 r5 Q# C0 FXenografts G( v% M7 h6 i$ m; W
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning# }: v5 }) P' _: I+ ~
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties" A8 f% m5 {6 `. i& p4 @5 ~2 f
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D$ |& M4 T8 U0 \
Cell Migration
) j% M" c3 t3 ^, k# E19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
+ E- g* U' Z3 K+ Q# ?" x1 u1 \+ J13. Chapter 11 In Vitro Reconstitution3 S1 @! t) e3 @; V% s
1. Introduction
. }: r0 K( g( T7 {2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems1 d5 y: `: H# M
3. Unit 11.2 In Vitro Translation y1 `; s) R+ _0 a( N! a/ x
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
* _: @: @" c# _8 R5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes% Y& H/ `" v: i+ w
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication# g5 e Y1 X4 B3 d- m
7. Unit 11.6 In Vitro Transcription
I$ r- R! }8 c) {+ n8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells9 U" u8 y# e6 ~' Z# E
9. Unit 11.8 In Vitro Translation Using HeLa Extract
: D% \6 Q1 i+ w2 Q( W7 m2 ?10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems' v3 z$ S% G! D! i
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 |* _% p" o' y+ ~- k
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
e" A3 ~' C4 P" r0 S13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
1 ]5 q% o* e# E" }0 H% s/ N- N14. Unit 11.13 Mitotic Spindle Assembly In Vitro/ T) W* g1 a3 q$ \' n' z
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes" z! _2 t& `/ y/ [
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import& T( e1 [4 O! W1 i% G9 D. X8 J% h
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import8 d3 m" x6 R1 N% w
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts0 S" ^- o3 s8 j: k
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
/ O9 p( j& G, Q20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
- A- J& a$ V! E) p- d8 x! G14. Chapter 12 Cell Motility
5 N; l4 V5 a k- p/ D% d( j1. Introduction) Z9 f/ R y, l7 @. U( e
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
+ A. W! U) ^$ F0 B; ^! f2 a t3. Unit 12.2 Invasion Assays7 T# V0 ?, z. t# }: I; Z9 _$ M
4. Unit 12.3 Cell Traction
, q$ v' o* X+ B4 e3 Q0 M6 h5. Unit 12.4 Cell Wound Assays
) K0 x+ J1 j6 V# H( V- ^( g, R6. Unit 12.5 Dictyostelium Cell Dynamics/ t4 j9 h, I* c$ U
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
6 L7 _& `/ d4 [- B! s8. Unit 12.7 Actin-Based Motility Assay
' s" e" p4 K. u3 H& q9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP/ \2 I$ y5 K6 w
15. Chapter 13 Organelle Motility
9 z* q8 W# L+ L1. Introduction
% K& ] x) S' K1 I. D2. Unit 13.1 Microtubule/Organelle Motility Assays
& {' J- Q; w* C3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin' |8 C% M5 f) @
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP9 H7 L; s3 i. e2 b e ~) u9 K
5. Unit 13.4 Movement of Nuclei, M# z* q: _; Z: Z5 X2 U/ z0 g
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching: Y% Y: y- {2 G' ?9 J5 j' }% k
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly* j Q0 ~& ~2 z1 {- B7 |2 C( ~3 l
16. Chapter 14 Signal Transduction: Protein Phosphorylation! n& U. A" ~, H6 {" m
1. Introduction
3 \( t* b5 |2 A6 i/ ^* G2. Unit 14.1 Overview of Protein Phosphorylation1 p. H4 U0 M9 y: f# S
3. Unit 14.2 Immunological Detection of Phosphorylation$ J$ V; U, I; R! U: h0 n
4. Unit 14.3 The Detection of MAPK Signaling4 F: L7 G" G6 _# h$ |( G
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation/ Z/ h; I$ k: k& a' u$ Q) t7 j9 O8 d
6. Unit 14.5 Phosphoamino Acid Analysis
8 N7 e b- S, x) r, m3 e) f7 }+ p) q7. Unit 14.6 Determination of Akt/PKB Signaling2 |7 N- q9 {! ~; n! a9 J0 h4 |) G% U
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
+ r3 L* @: ]3 }8 {$ ^9. Unit 14.8 Rho GTPase Activation Assays5 |& _% g# N: s5 K* B' z
10. Unit 14.9 In Vitro GEF and GAP Assays9 b, a, O v- k' f! X+ j% f; F& `7 E
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
0 v! T7 x- w d+ L0 o* Y6 U3 _% TEnergy Transfer (FRET)- {( ~. G0 O) w% Q
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells1 S+ }7 G! i j( v, d& G
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
9 r; L! l5 N$ L$ b8 T9 R3 n17. Chapter 15 Protein Trafficking9 d* [; O) U5 X j8 b
1. Introduction
7 J3 M M2 v4 `; P2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
6 c' |3 H, z7 G+ K3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
( w [8 k5 ]2 G4. Unit 15.3 Endocytosis: Biochemical Analyses
* P j; d8 z7 x$ U5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
: `+ g7 P- O) k2 u& @( v6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells* f& ]$ e) ~. f1 q$ B- }
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum& X* u1 G/ N3 N4 t. t1 K
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
5 j$ R. x3 o& \2 }9. Unit 15.8 Analysis of Protein Transport to Lysosomes7 g% |; K' ]+ _7 z' r5 c% L
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
1 x3 f. b: r0 I$ R: X3 o# U11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
" P8 J% Q- |2 m( {4 l12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
9 a v8 N# ~; M13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
: M, D. W8 `$ u4 H: i+ h2 h14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
0 z# L6 D. U4 A6 C- e15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
$ ]/ n! j) y- N, x& \' J3 @0 ^EGF Receptor1 e$ x% g; d, x. `) ~9 b$ a+ M3 X
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers. y8 ^5 N9 ~! b' A! C
18. Chapter 16 Antibodies as Cell Biological Tools% E! E) h% J6 L2 Q& H
1. Introduction
3 s. [8 o r0 F9 ?2. Unit 16.1 Production of Monoclonal Antibodies
% j# p0 M( f+ `' [" }3. Unit 16.2 Production of Polyclonal Antisera
9 t& i# g: l7 n* g( k1 x4. Unit 16.3 Purification of Immunoglobulin G- H. [( _6 l3 p9 B" P
5. Unit 16.4 Fragmentation of Immunoglobulin G4 ^* v! I; Q3 q- S8 _
6. Unit 16.5 Antibody Conjugates for Cell Biology
1 f; }9 c3 T9 `2 S2 t7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
2 T8 X3 m0 {2 f. ^% t6 G1 y5 u19. Chapter 17 Macromolecular Interactions in Cells
$ i9 ~- @ x+ d' H/ w8 ^+ Y1. Introduction
! [' s, V# B r2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)# }" F1 h0 B: d5 w' }
Microscopy
, h1 d+ @) }- y/ ~; ?5 ?9 u3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
C! J. `- t: M- p4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins* o# S0 j. m4 @$ r% m( Z; T
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
7 f5 w. ^6 i! e* H6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry9 Y/ k6 t+ M" a/ g" r+ s
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors: h% r, {2 Y9 ]% U3 n
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
' N$ T3 N( |) Y& M! Z7 h+ xGenomic Sequences In Vivo
- s# M2 I8 X( w: r: w; f( ?# i* n9. Unit 17.8 Isothermal Titration Calorimetry8 `7 E# x, b) A/ i+ N# B
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells( t0 R* T/ G" Y9 E
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
% w9 m- L! g2 r: P9 l+ d12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein1 |6 h, T5 r Y* Y. B+ u; c
Interactions (RFAP) in Live Bacterial Cells! a2 T0 R7 }5 o; v. V( ?
20. Chapter 18 Cellular Aging and Death
$ T9 f3 A! |) }1. Introduction5 ^6 ]! }5 Y; \( O2 A" X: k
2. Unit 18.1 Current Concepts in Cell Death
L) A2 U; z4 K$ f3 I1 k3. Unit 18.2 Analysis of Caspase Activation During Apoptosis. E& K1 m! Z4 l+ t
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
3 Z1 M" k, |; I6 a4 {) C5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
" N1 S6 U! P3 r6 o6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death4 Z" }- \3 z+ A, T
7. Unit 18.6 Analysis of Telomeres and Telomerase5 Y# a$ X7 h' `1 c! j7 _: I
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of5 @; h- F4 G* i
Poly(ADP-Ribose) Polymerase# B' Y- w0 q5 U' W- U
9. Unit 18.8 Flow Cytometry of Apoptosis* Z" E6 O+ E' X: a; i& v
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase' x/ t' Y. P% q% x0 A; [. s9 G
Assay# ~4 |$ Q7 q0 q z; o, f$ U
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
* @0 _ O6 i0 [, z7 ^21. Chapter 19 Whole Organism and Tissue Analysis8 k) c2 B) t( m* j* @9 A% S5 V
1. Introduction( `2 C$ H) g/ a R) i' ^( o
2. Unit 19.1 Overview of Metastasis Assays2 o& e% i( Y3 n. W& W
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
& Y. a N ~1 _) C4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
% T, A. j! K6 r; L( DExpression After High-Fidelity T7-Based RNA Amplification
C1 Q$ y; p* s, L* Z5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA, E$ v0 L- H: O
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model. J, F8 ]/ I7 J0 I' Q
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
- Q, {1 O( C$ d( b8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
+ |$ ^" @$ a1 M3 ?8 \& E' B4 _: Z9. Unit 19.8 Embryonic Organ Culture
/ o% y5 B6 c7 t9 s6 O10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin2 ], x$ Y8 [* K/ X0 T
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
( _& ~+ z! A* n+ x% L. ?12. Unit 19.11 Generation of Transgenic Mice
1 C5 k3 Z1 c2 f! K2 G: l. ?. d* f0 |13. Unit 19.12 Overview: Generation of Gene Knockout Mice
* y0 G1 W! a& G1 n' S3 }# p4 g14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production2 S; l$ A# F: L
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
3 `# j0 [* `* \22. Chapter 20 Expression and Introduction of Macromolecules into Cells) G/ l/ f& T/ z9 Q+ o6 R
1. Introduction6 f/ M+ \# v8 K& [) i! w9 W
2. Unit 20.1 Direct Introduction of Molecules into Cells6 l" s5 a; y. \% Z0 F9 R/ V9 f
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System( E2 a; ^' G, k4 j5 T
4. Unit 20.3 Calcium Phosphate Transfection
5 o! n' z: @' t- I' {3 L! e6 r5. Unit 20.4 Transfection Using DEAE-Dextran
2 i+ [* ^: R6 I! ?8 ~- B6. Unit 20.5 Transfection by Electroporation- X8 L3 E- w0 ~: j
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents/ `# G6 W. x8 P) j7 r e
8. Unit 20.7 Optimization of Transfection, M. H$ J( ^, t" u& e7 Y `
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System9 V3 S( B, L! z. y' _
23. Chapter 21 Fluorescent Protein Technology' t; z# `# Q' i$ S
1. Introduction
8 K; F$ w4 x( ~9 s- L& [2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
- b3 |. A8 o1 i- Z( g8 q3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)+ l% y( y3 s2 j" h/ L) x
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
4 Q% N/ W* ]/ i1 NComplementation (BiFC) Analysis
) w2 f/ }% n. ?5 a' {* t5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
3 {% x+ m7 I- s7 ^3 A( Y; S4 Z7 _6. Unit 21.5 The Fluorescent Protein Color Palette
b0 Z- h2 v$ v/ O) y8 T7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
' ^% H- E* z8 C' C8 w24. Chapter 22 Cell Biology of Chromosomes and Nuclei
, L4 Y n% H. a$ @, M7 A8 F1 n G1. Introduction1 w8 A6 h6 U# V% K4 G l7 |" ]3 g
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis% g( V9 @- D- e3 C
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
4 S2 b! v/ u1 G& p% U4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
! B' ]# `' e. h# Y2 ~) y$ ?8 T8 w m. q4 P5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
, W5 u* Y# S: o/ _6. Unit 22.5 Multi-Color FISH Techniques
8 F* i" L6 W4 d0 C. i$ l2 A4 J. l7. Unit 22.6 Comparative Genomic Hybridization
1 i& p; l8 h6 s0 y3 K, t8. Unit 22.7 Sister Chromatid Exchange
( k4 [* m p$ n" l0 e. T9 k9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery6 L: T0 y# U6 ?1 V
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
, c5 c& W2 f1 c11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
+ K7 C- i" i" i! i& g J9 z; E12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
( d; }6 s. ^& p: n- k# X) v13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
: w7 S; H: j! d. q14. Unit 22.13 Monitoring mRNA Export5 _. J1 L7 V# h+ b% d
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-+ o( S. @8 A4 \7 a( V9 N+ m
Field Gel Electrophoresis
. T2 ~+ E* _3 R' Q3 p25. Chapter 23 Stem Cells
' ]* [6 c, F( J* U. J7 R1. Introduction
: t) I% p5 _" t# a/ L* T5 L2. Unit 23.1 Stem Cells: An Overview b) @1 D/ {3 k r2 T2 V
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
2 O: s/ r. @* V+ D+ H2 f) Eand Differentiation as Embryoid Bodies
# V! X3 H8 |. n9 Y2 F9 R4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood% M# t0 _/ g( Z P. ]* Q
Vessels: b" j. G9 H; E6 k
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
# D; |& z7 G" p6 R8 [# OAdipocytes
5 G* e6 U% r5 F7 r6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
% e- ~" n4 s: z% m+ d2 U* Q. N7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells M3 j% v% ]/ ^4 u
8. Unit 23.7 Neural Differentiation of Human ES Cells8 f H; C4 {" ~0 J
26. Chapter 24 Lipids0 T H1 w: A' [
1. Introduction
" W1 n; |; Q- L& N9 }2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking; w' l7 b" p8 W7 l
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
* u5 D* p/ E5 [1 x" Y4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
2 ^1 V# ~/ m0 I+ @. L6 R. T* T5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
% p) F j% ~, q( `27. Chapter 25 Nanotechnology1 ]* M: d5 Q& z% G9 s# V
1. Introduction
% V" M8 u6 P8 z3 o! K! }2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
4 @) e9 j7 k" Y- k! h1 N2 _3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering6 A. i# G7 c) r, {( x% Z, C
28. Chapter 26 Viruses! n$ \$ y8 e- d8 }8 a9 K
1. Introduction
5 ^! m9 a+ X+ \5 f, y, G2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
( C/ C5 \7 d7 [3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
- _! }; U5 ~5 B0 e- y4 OCell Entry
* ]9 V$ }" U! _. B9 d4. Unit 26.3 Methods Used to Study Respiratory Virus Infection5 c2 T% F$ z. Y* n& Z- I6 r4 O
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
$ b: R, S4 w% I8 E6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
" E4 s4 J6 B8 r29. Chapter 26 Lipids
9 U! [2 M3 P+ N1 K$ p& O7 O/ s1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by5 @) b1 f7 s9 J- \
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
$ `- R2 m. `7 @$ `! J: M1 G- \30. Chapter 27 RNA-Based Methods in Cell Biology
+ i8 ]2 U( z% }2 E! w! o1. Introduction
" t8 R9 O; L A+ w- x+ S2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
3 z7 b- a9 b* O) b, L/ w& l/ @3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible2 Y1 t/ ^. _- C. Q- h6 z
Rescue in Mammalian Cells. b1 D3 [ {9 R& ^0 f2 k" h
31. Appendix 1 Useful Information and Data
( M) u3 L' o, a5 C1. 1A Useful Measurements and Data& b- W$ g( K* r+ H( Q' O
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research( ^3 P0 Y" T4 _ D) U9 n7 e( o
3. 1C Identification of Motifs in Protein Sequences6 r( K9 j" k" m1 Q
4. 1D Safe Use of Radioisotopes8 J/ v; ]0 }" Z! ^
5. 1E Absorption and Emission Maxima for Common Fluorophores
% F- M% Y; p5 r& ^6. 1F Importing Biological Materials: Z# k$ r% U* P
7. 1G Centrifuges and Rotors2 ~% S* J7 c: j
8. 1H Internet Basics for Biologists
$ O3 n: Z# y" `1 G) h t6 v6 S32. Appendix 2 Laboratory Stock Solutions and Equipment
, s, ^5 Y! M0 k D9 M1. 2A Common Stock Solutions, Buffers, and Media
$ ~& c5 D" Q& m" N# Y2. 2B Medium Formulations; D" }% f! S7 L6 S+ c
3. 2C Standard Laboratory Equipment
2 f% y. m5 m1 j, c0 B8 B' O33. Appendix 3 Commonly Used Techniques
, L3 R1 m+ N7 c% K1. 3A Molecular Biology Techniques
* J# }" w, h8 z4 V6 y7 ^2 V; ]2. 3B Spectrophotometric Determination of Protein Concentration
- }0 K8 }+ ~' @- U& p) R. N% b3. 3C Dialysis and Concentration of Protein Solutions; D4 q. w" U' k+ ~) _& ]# @- }
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
# k5 v$ M8 I- p! k1 H1 N' ~4 `5. 3E Silanizing Glassware1 L* B+ w) F/ B8 q; g: k6 _
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
3 j/ i# a: V# z8 Y" |7. 3G Micro RT-PCR
( G+ H- f) ]& p5 \8. 3H The Colorimetric Detection and Quantitation of Total Protein
: M6 [: I) W( B34. Appendix Suppliers# d* s; D( F/ a9 u6 A$ \4 k
1. Selected Suppliers of Reagents and Equipment
, V7 |6 `5 @# ]
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发表于 2011-3-8 15:53
