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+ u( }5 Z9 J6 [! O* HCurrent Protocols in Cell Biology 2010年完整版 5483页* f6 _: f) `/ L5 x# b
8 W6 R$ n, ?- [- [Online ISBN: 9780471143031
3 |% h) h K4 S5 HDOI: 10.1002/04711430302 i3 h8 X( f5 ~9 T. I! y7 D0 _
: b% S$ _1 K' E1 |$ _Table of Contents
7 Y( @0 o$ p' o6 Y0 X5 P" Z) |1. Preface* E# v/ z% w; B7 Z$ P4 w
2. Foreword+ j( p) D! m+ N+ N* H3 B: M; u
3. Chapter 1 Cell Culture) z9 d. e" G& h, j% w
1. Introduction$ s/ x/ J; K4 X4 A3 i& L; k
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture/ W7 j! s( ~7 n$ M0 Z( f/ S
3. Unit 1.2 Media for Culture of Mammalian Cells z! s' Y/ _9 `; \ F+ [+ [
4. Unit 1.3 Aseptic Technique for Cell Culture# C6 R% Q* h% u1 s
5. Unit 1.4 Sterilization and Filtration" c5 f2 d. X+ V# ?
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures9 p8 A2 g( [ C: C- g( ?
7. Unit 1.6 Media and Culture of Yeast$ d# c' R- ?+ k/ T
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging3 M- f m0 c- [8 p! [5 z
4. Chapter 2 Preparation and Isolation of Cells
; |0 B" _" I: T4 P7 w7 }1. Introduction) k8 N- f. `1 u3 |/ K# F
2. Unit 2.1 Establishment of Fibroblast Cultures
7 z3 Q' d7 y6 K) c3. Unit 2.2 Preparation and Culture of Human Lymphocytes
4 Q$ n& F2 z, }) Z4. Unit 2.3 Preparation of Endothelial Cells/ {5 B3 A6 e: O- t1 A, A8 d
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
- D) m4 h: @; Z6. Unit 2.5 Laser Capture Microdissection
% x$ B' k0 y% q1 V$ J6 }: Q3 c7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures$ n; y8 l5 T% ~2 J7 G- X
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells \; A& [! Y' q- s
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
) D2 ^" A+ W8 S. f1. Introduction6 A) T( F- Z0 w
2. Introduction3 |; I/ B3 k, n/ ]3 Y$ F: x
3. Unit 3.1 Overview of Cell Fractionation
4 T# w) [$ w% e2 v9 J4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains& o: ?. d" O5 @5 u' f
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
' ?4 S' n6 }. C! i0 y+ a/ b6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation- S; n1 A# W1 g. \1 r& b
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient5 i( z: u0 [: ]: ?3 v! r+ V' T$ B
Centrifugation5 C! c+ a. K6 p+ e/ H
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient4 c# ~7 t% z1 H
Centrifugation# A( _% ]9 K4 X9 B
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
) ^- C- J6 P* U# \1 h% A( r: r( u10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
8 d% y/ v7 K0 S11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient3 C0 B. m) y$ k8 T' z1 h
Centrifugation
w. A6 a% Z( S12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues5 o' L! g4 g0 [" M0 ^3 r
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes0 Z' Q) Y0 ]/ X1 x; J
14. Unit 3.12 Isolation of Synaptic Vesicles4 \3 a) J6 b! X& [/ ^& u, m! q
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
! A, E1 q! x! S P7 B16. Unit 3.14 Isolation of Melanosomes
: H$ @3 g9 V5 W) ?0 B17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation! _! f4 i/ ^9 M
18. Unit 3.16 Isolation of Mast Cell Granules7 Q" S, I( H4 z) K7 P% A& s7 f
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
5 h7 O3 G+ j4 Z# H& q9 t1 N( p20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
$ Q2 I( Y2 `! N5 |7 Q8 t; ^9 X21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
- o* ^1 _( x! Q8 U: {0 J8 @0 T22. Unit 3.20 Isolation of GLUT4 Storage Vesicles$ O/ \+ y( T& ^! Z% t
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
/ c1 p5 H6 n' h5 C& U24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
6 s% m6 r3 W1 k5 t1 c+ j( W! w. NFluids
; [; @) t5 k$ n$ U- b. y ?" b25. Unit 3.23 Isolation of Intermediate Filaments T. D$ Y: l y1 }$ F
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle
1 H# l% x' Y @, ?27. Unit 3.25 Isolation of Myelin
3 l r" ?! _5 ?( Q7 u" M28. Unit 3.26 Isolation of Renal Brush Borders9 n9 N+ L' T {9 n; n% f
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane% U6 T Q; O d
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts4 \7 f3 C6 J7 g, j! G
30. Unit 3.28 Isolation of Amyloplasts
5 S+ a" M. \/ a2 R: q* H31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins1 X8 Y$ ^6 S: c4 J! {4 M( t
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
8 H3 ]: l$ E$ kCentrifugation3 M* l" Z4 H( x* Z( H% @* L( T
33. Unit 3.31 Isolation of Neuromelanin Granules
1 W Y; @2 {4 H8 C4 s8 U34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
8 {8 X0 _4 z, |8 EDensity Gradient Centrifugation0 i6 z( [2 `2 [# h4 b6 H8 I9 s: p
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
- ~. P3 }# J" w$ Q0 D5 c1 q36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation. y( c0 Q. W! ~, |+ Z8 L+ v) N
37. Unit 3.35 Isolation of Platelet Granules
; K4 f& ^9 O/ |% j" x0 A4 T38. Unit 3.36 Isolation of Nucleoli! c5 K% R# [8 Q! Z4 x0 b0 H
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
% P+ J4 r& u5 R3 z* H40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates2 _$ a# J" E9 O9 @. i2 t7 a
41. Unit 3.39 Isolation of Chromaffin Granules4 ^8 F& R7 v: p# j% L, Y, U' u
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
3 [. L, G1 H) T5 |- V% L6. Chapter 4 Microscopy3 B4 B& ?$ M8 i1 }" H0 m
1. Introduction
: y6 H& l7 c8 C! x2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope9 ~3 f. M/ r) l' p" n( K7 i
3. Unit 4.2 Fluorescence Microscopy3 r* B1 h# b( W! C
4. Unit 4.3 Immunofluorescence Staining3 B+ V( d0 ^; m/ |
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
( l7 p2 P& H. {6. Unit 4.5 Basic Confocal Microscopy/ G' o" l3 W/ _ l
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
* c+ o; w! p5 L0 `" }5 }# x( G8. Unit 4.7 Cryo-Immunogold Electron Microscopy! T) G, f) H* V: r+ b6 }! R, r
9. Unit 4.8 Correlative Video Light/Electron Microscopy
$ v6 g- |( X8 t6 D7 w10. Unit 4.9 Polarization Microscopy! X6 A$ }6 ]& q" b+ q
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
' B; v. C+ Q4 g" v. |12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues0 U1 i, i6 u% n7 X0 O1 i
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface8 q, B, h2 S- q! p
Events
; e d) ?7 }9 J" Y14. Unit 4.13 Fluorescent Labeling of Yeast
3 b! g/ b- ~. T- g$ n# J' U15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
# [( i4 c6 c" U16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy* p% {& H4 V5 a! W/ e K' x* F+ n- \# `% o$ S
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules' B# n9 ^+ I& [, s6 b! k- n
in Endocytosis
$ x1 @. h9 s# I& z' h8 g2 J18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
) w9 j6 g0 [$ t) ]5 V6 X19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development' l8 J4 d4 C" w6 \' W6 |' _2 A6 R
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images! ]' L7 \4 p$ f! M" B+ k
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions. v0 a: p/ c& I, b7 V, W
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
( |9 c% f% i. e, f+ X23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics4 C: B+ j [, D
24. Unit 4.23 Interference Reflection Microscopy
5 |: X) Y; p7 a( a25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach# f, ~/ X* H( C1 u* f f+ h
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches/ a- M2 t; z: y5 m5 ~& h
27. Unit 4A Organelle Atlas: Appendix to Chapter 4
9 M( G- ?* k/ E% o: N& O- ]6 r$ D7. Chapter 5 Characterization of Cellular Proteins+ Z. e& ?' w5 A. Z& u
1. Introduction
1 q6 g8 C/ j5 b) o! Q1 Z2. Unit 5.1 Overview of the Physical State of Proteins Within Cells) u+ K' z" u4 z5 A
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
! J% Z: }' T L0 Y; `4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients- h) C, S8 g& J: n- f3 |5 c7 f
5. Unit 5.4 Analysis of the Association of Proteins with Membranes, L! K/ W4 y- H
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration); F9 w3 u+ `' O( j
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass; F1 u" x3 C5 V0 n9 f
Spectrometry
& c3 e6 E; t$ m8 b/ L8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening% i$ g$ T5 C5 Z7 @" C) d3 \
Applications
& r& c/ h } b8. Chapter 6 Electrophoresis and Immunoblotting+ o" E# R+ p+ g9 l- U, l2 D
1. Introduction
; X5 F4 }4 {# R: y6 D( X2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins% t$ M8 P; D s( y: B
3. Unit 6.2 Immunoblotting and Immunodetection
/ ?9 m1 p3 B' T4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
R& o/ I/ z3 z% d* O0 M5. Unit 6.4 Two-Dimensional Gel Electrophoresis
6 g; e' Z9 k* c6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions2 u) B1 }7 }+ L( _
7. Unit 6.6 Staining Proteins in Gels
( E- g- D6 T2 C- ?) G0 b9 p8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
: u3 b# m! {$ I" j3 I# P, n9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
3 F% ^/ b5 O) ^/ _; l: W10. Unit 6.9 Digital Electrophoresis Analysis5 c3 `, E( p8 _5 M
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
q* ^9 i2 ^6 o( H12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
6 ]: g7 W) |' }; N; i7 aSingle Cell Gel Electrophoresis (Comet Assay)$ q# X8 d1 P# @6 l& a8 ~+ V7 }
9. Chapter 7 Protein Labeling and Immunoprecipitation
3 \4 ~7 L h7 B4 N6 A& h1. Introduction7 ~7 q3 M4 J/ O- C
2. Unit 7.1 Metabolic Labeling with Amino Acids* a8 x' q9 N9 l6 L
3. Unit 7.2 Immunoprecipitation2 H3 x3 p5 { v
4. Unit 7.3 Metabolic Labeling with Sulfate
8 ]9 m2 I( \ y5. Unit 7.4 Metabolic Labeling with Fatty Acids
! B2 X$ d2 }6 o6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
, k7 [5 ?/ a5 Z& G7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins, Z) O' o, |; h7 C/ v
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
Y4 k& w0 A$ s. W# |9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars# _# M* C9 r, H* j! J' Q
10. Unit 7.9 Analysis of Oxidative Modification of Proteins6 p9 `$ C% U# N8 i q& D" V
11. Unit 7.10 Radioiodination of Cellular Proteins
$ N+ N" H7 `/ l0 \4 i10. Chapter 8 Cell Cycle Analysis3 T3 j- k- W1 }. l
1. Introduction0 ?9 w) [8 s# U5 E: ]! J
2. Unit 8.1 Overview of the Cell Cycle
5 t2 o. K% a# i( U5 C7 j1 {( @3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle: F$ O L" Q1 U; b* _+ c
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
- I/ }) R! X1 j5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry7 A0 r/ I! i% [8 T J
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells& [) W" g* S9 |3 C+ h, E, `
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
8 O8 n. ~8 ?4 O: X11. Chapter 9 Cell Adhesion; u- _" A, M# G- V6 _! @
1. Introduction
1 L% P! Y9 R$ j" G! f2. Unit 9.1 Cell-Substrate Adhesion Assays9 V8 z9 g" l) ~
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force' R1 ]; c+ M+ W$ l8 a/ u1 l
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion1 t/ e& H/ j) L2 B
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion% k, ~1 Z6 H: e2 \
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules/ D. e W' O p* U/ J% o9 I
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
y( v9 f( j% p) N: l12. Chapter 10 Extracellular Matrix
9 M4 R0 i* v+ T1. Introduction
$ r( Q2 t" ~ ?& P) t2. Unit 10.1 Overview of Extracellular Matrix) a; d8 L n# |/ h' g3 u
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors1 P( ?" c) n2 F. g3 {# X7 Z" ~
4. Unit 10.3 Preparation of Gelled Substrates
( C; Z/ {, O% C& |5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
1 E9 E) f, h* w; z2 ]Endodermal Cells4 C7 s+ l- f& j, k1 j* o
6. Unit 10.5 Purification of Fibronectin9 k7 k1 }! f% g% ]
7. Unit 10.6 Purification of Vitronectin; \2 F$ _- x+ B# {# X+ d
8. Unit 10.7 Proteoglycan Isolation and Analysis) {7 I3 ]3 Y+ b8 }7 J* p. {
9. Unit 10.8 Matrix Metalloproteinases
8 ~- j! i8 i3 j0 P6 j10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
0 b, {/ s+ C1 I8 Z: R, E* X* j11. Unit 10.10 Purification and Analysis of Thrombospondin-1; ^3 K* X+ S. y0 p7 k5 C& y
12. Unit 10.11 Purification of SPARC/Osteonectin
9 s A' i z2 G' U13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
* T" E( {. x3 I- ]8 r14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
* L7 l* u" G6 H15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor1 n$ U, m8 N: s% A- q, `
Xenografts0 J- z8 S f) l7 Z) m) \. A
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning4 w2 p( ~5 W( d" B$ U) i1 m* P
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
. O6 O7 j- W$ N18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
/ \# H1 k: S9 [' aCell Migration2 A3 Q; x# x& D: ~" j: ?
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
0 l! W$ g: `( \( _$ o* P9 p: \- p13. Chapter 11 In Vitro Reconstitution7 y2 J. L( T, Q# H
1. Introduction
, Y! i* F8 i( h# D2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
( P3 K2 a" L% X$ {3. Unit 11.2 In Vitro Translation
+ N5 u1 h6 X; t* @4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
- X8 L8 y! \1 n# e3 m$ I3 S5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
, D1 U7 T- a# h* `- M6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
6 O; s" B5 Z' v2 L- z) I5 E: w4 c7. Unit 11.6 In Vitro Transcription: C: ~( z% v; K( Z* O
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells1 ]6 F: Q! U |; _; b; y
9. Unit 11.8 In Vitro Translation Using HeLa Extract0 }7 O2 J& S6 \/ g/ U5 q" c
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems3 z" f% z/ B- T6 O0 o
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
/ n. r, Z q0 |: @0 \! s% L) Z" q. v12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
3 \$ Z5 A) v3 q. C. i& H: \; A13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts9 Z, {+ O3 d( a2 J& b7 Z) q
14. Unit 11.13 Mitotic Spindle Assembly In Vitro: f7 @: B$ | ?- O( R' `
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes/ r. K J y" L% s' }# z
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import0 z* y" \/ G, y0 c4 J3 ^
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import5 |2 ^/ v" L$ x' G
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
5 U7 _) M+ a8 [# a6 N19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells9 k/ i$ R2 T! X7 o8 s5 |+ {+ Z
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import; U( j' p( ]4 q3 x2 v$ ~5 w- C a
14. Chapter 12 Cell Motility
( [5 i* h6 b0 z: z1. Introduction
+ k5 g5 u* k; b0 A" o$ j1 A1 B, c2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
) q: P6 g+ n* y" ?1 ~1 L8 |3. Unit 12.2 Invasion Assays* S" n6 {# U2 ?" n
4. Unit 12.3 Cell Traction' A" f4 O$ q4 t( X0 R7 _+ K
5. Unit 12.4 Cell Wound Assays7 f6 r% R0 _. N! ?; {0 j
6. Unit 12.5 Dictyostelium Cell Dynamics
9 r8 J# h6 z4 @* G! ], O. S7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils9 E* t) Z, z u; F* o$ @
8. Unit 12.7 Actin-Based Motility Assay9 K- \( F+ R) A0 ^& p6 m! k
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP" ]1 l" x( A0 `' }1 T
15. Chapter 13 Organelle Motility* s4 T( z+ I# C/ R8 w2 Z2 y/ \
1. Introduction1 q- P$ R u' u$ N( o% c. G" E( W
2. Unit 13.1 Microtubule/Organelle Motility Assays: U" o, w+ g# ]' g5 J2 X1 S
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
( P2 T2 r* z( x5 A/ h V4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
' w" y+ k+ @3 k/ U8 Z5. Unit 13.4 Movement of Nuclei9 A5 ^0 C1 O. K( y
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
, A% l+ d3 A8 [3 P7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly* r' }+ D' s% I D w
16. Chapter 14 Signal Transduction: Protein Phosphorylation
; `* L8 Y! P, s+ k1. Introduction& c3 r3 ^) l4 }8 c- q9 K) K$ I9 V
2. Unit 14.1 Overview of Protein Phosphorylation
% @) d; ?7 H0 S- g! X( `3. Unit 14.2 Immunological Detection of Phosphorylation& P$ I) X. E c6 p2 \: A
4. Unit 14.3 The Detection of MAPK Signaling
' j: H; z. q# P a; p0 Y$ }5 k" m. f5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation3 {% F- A9 I& }: L! g: R
6. Unit 14.5 Phosphoamino Acid Analysis
# ]% ?, e# g9 R% U- t% @7 ^( h2 R! C* n7. Unit 14.6 Determination of Akt/PKB Signaling* [( f( g. H9 l" q/ c$ P- \; E
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
7 @2 H: I2 Q Z3 ]. L; n9. Unit 14.8 Rho GTPase Activation Assays& I. m( |% a8 t3 S9 u$ ]
10. Unit 14.9 In Vitro GEF and GAP Assays
+ T# M! e+ X& x2 m5 d( {9 o' K9 q# P11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance9 ]0 _7 t2 b# o* p5 T) C
Energy Transfer (FRET)
% {2 }. E" ~, q! J+ z12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells$ {( g# l$ T1 K6 J
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
) E; |6 V$ }6 b/ l17. Chapter 15 Protein Trafficking
, \6 a. k% O, ?2 E: ?+ T# K- j4 V, {1. Introduction% i/ e, k! b4 n1 v# k' c/ m
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
, v$ D6 N2 v9 n+ I. T* I& a& y3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
7 X$ ~& D) M% j0 O; A3 o4 R4. Unit 15.3 Endocytosis: Biochemical Analyses$ ?; P# A0 j: J1 j! z$ d
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
: F$ z& u2 L( g5 }' E7 v' R Y6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
2 w+ w4 [5 x2 @% r7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum# n5 w# y& |$ b* G
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
( `6 O: P* Q' J8 W' ]9. Unit 15.8 Analysis of Protein Transport to Lysosomes$ X% h. T: m" f
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
& ] J* o) M1 p# E11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network) d$ k0 ], I+ l' e6 \5 O
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
. ~! {& _/ _- Z! k& P, y( r; ^9 f* G13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
' i5 g1 N$ Y4 _14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
6 {& T+ \: O( }3 P8 m3 V15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and$ b& Y" a: T4 i( Z4 C0 I8 v
EGF Receptor) |0 r: D1 U, j* w2 |7 m2 y! t
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers& j0 ~4 ~1 G: u0 o) h& \
18. Chapter 16 Antibodies as Cell Biological Tools
$ G6 h$ r" K. {* n9 C1. Introduction
+ I' R" r4 @: n, w: a; k5 ?2. Unit 16.1 Production of Monoclonal Antibodies
6 W% S* C7 r1 _3. Unit 16.2 Production of Polyclonal Antisera3 ?9 M; R2 k2 H3 E9 A! }* J
4. Unit 16.3 Purification of Immunoglobulin G
% T0 |+ ?& q5 z/ L1 d) m& c5. Unit 16.4 Fragmentation of Immunoglobulin G
0 p7 K! Y% E! D6. Unit 16.5 Antibody Conjugates for Cell Biology1 G8 u- d* @& G& W' m$ b
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
+ d7 h9 P8 U. d. C. z" J19. Chapter 17 Macromolecular Interactions in Cells' Q3 l# H3 ^4 [# X0 u
1. Introduction7 {0 p) l! J1 l4 @( ~+ o
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
; d3 B# D- ]: E) C3 t3 k# kMicroscopy" |2 s5 _7 s; E+ p* g# O- x
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
/ p( d7 O! U" h4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins% z( b3 a7 |- h6 n3 @' w/ [3 H
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries3 y4 k7 ~* B: I; V; S
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry& [) F, K! a0 P9 [4 k* @7 G
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors+ t. a! v' `* x8 a- p0 o6 X
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
" t* Z+ K8 B4 f3 i% Z( S" JGenomic Sequences In Vivo
: o5 F, J) G, u d: f5 t V8 D9. Unit 17.8 Isothermal Titration Calorimetry( I8 L( }0 f( d$ Z
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells6 ^- ~# Z% e; B
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking+ K9 p3 i6 [" r
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein6 N6 p3 q6 k. L( z" p0 j0 n1 y
Interactions (RFAP) in Live Bacterial Cells
) C+ O: s, s" q8 M" e20. Chapter 18 Cellular Aging and Death
3 A$ X$ x ~8 `; i2 k7 x1. Introduction/ P' Q' T3 K9 t
2. Unit 18.1 Current Concepts in Cell Death5 d, \, J& Y7 Y9 r
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
) p5 V) E: f$ I( I2 x4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
; L5 D1 U- j- {: N5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)5 x& @3 y8 ?2 L0 k1 ?8 D: j
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death* a1 P2 T) I$ q+ |& m
7. Unit 18.6 Analysis of Telomeres and Telomerase; \" R9 E. J) r' d' g
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
; m' ~$ V4 K$ V+ Q7 dPoly(ADP-Ribose) Polymerase/ k3 y$ o0 k `' u! y5 T+ W
9. Unit 18.8 Flow Cytometry of Apoptosis% B% D4 u/ U! f2 X9 g* U: @! {+ C
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase# i) @ a% w3 o
Assay
$ q B1 a5 u+ \5 e S11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
! O6 z2 X. [* g [* k3 S: P _5 T) c21. Chapter 19 Whole Organism and Tissue Analysis7 J$ _( W/ V! M0 n$ o* r$ p
1. Introduction
. m+ e1 t# F7 p, d0 L2. Unit 19.1 Overview of Metastasis Assays
) Z- O9 k# ?7 b) x5 X: }9 d% q3. Unit 19.2 Tail Vein Assay of Cancer Metastasis; P1 ]8 f% F9 r8 T$ L
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene, o5 L6 x! A T3 R* \
Expression After High-Fidelity T7-Based RNA Amplification; x2 D- @) D1 O" R- X/ f' `0 A
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
# v" _% n9 d. {9 }6 s8 {6 E( a6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model8 y$ V( ?% Z- ^( O4 Z/ k: m$ g
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
7 H$ u1 u4 _+ n- R* H8. Unit 19.7 Imaging Tumor Cell Movement In Vivo4 e! ]# u: V& f$ x
9. Unit 19.8 Embryonic Organ Culture
, n" @9 ~" Y9 a2 I! A& R1 C10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
) a5 N, B+ s x# C$ O/ m. L/ @" ~11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice5 m8 |) c( b2 P; m3 Y, j3 G8 | t
12. Unit 19.11 Generation of Transgenic Mice
/ V) f! l% u5 c; r c13. Unit 19.12 Overview: Generation of Gene Knockout Mice6 g7 C2 d; f) }+ [
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production0 `7 L8 m: S3 M+ ~) g+ A
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
7 x" N& @0 `5 Q" f( z1 {22. Chapter 20 Expression and Introduction of Macromolecules into Cells6 q5 u( z5 R, v+ u3 s v
1. Introduction( I' H3 W. E3 _5 B" ~1 s
2. Unit 20.1 Direct Introduction of Molecules into Cells" F! z% \! w! n! G( u5 G4 a
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
/ m0 V6 b) C4 H3 [- x5 |4. Unit 20.3 Calcium Phosphate Transfection
6 z# X" ]) W) y% [; i$ ~1 d5. Unit 20.4 Transfection Using DEAE-Dextran
+ P7 W9 p0 \! q6. Unit 20.5 Transfection by Electroporation. _0 H U- W! S3 a. _4 Q9 c2 d
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
' ^8 t5 o) \# b7 v1 B9 y8. Unit 20.7 Optimization of Transfection
* Y' T1 K4 B W3 @4 G! l2 O1 L' K9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
5 H6 L# c2 |, t. z23. Chapter 21 Fluorescent Protein Technology
: G5 U2 o$ }) R3 U- P& o2 Y1. Introduction- ^& W w; p; V8 z4 D' `
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
+ h/ j; @- R3 i% u" A# ?% ?3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP); B8 N3 z5 F3 g D8 y9 Y. B, i' H& g
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
: I6 n' w7 j- K' A, |: R' ~! ]Complementation (BiFC) Analysis$ e" Q+ M! U" `! @9 p
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology. D+ ~* v9 X" Y6 R- p7 z/ ]$ P, I
6. Unit 21.5 The Fluorescent Protein Color Palette
9 U0 v Z# ?1 m+ }$ g7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins: G m6 v+ ]0 h8 G
24. Chapter 22 Cell Biology of Chromosomes and Nuclei
0 a( V1 p/ {0 P1. Introduction
! d1 P6 a" S$ E D' ~% ^( y+ i2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis! |) c! d \2 d/ z: ]9 n0 d! Z! N
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
$ G+ }3 x% i; W( h# z5 \4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis9 ^: Q3 \7 X, r; X. R
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)) ]6 ]) m& n7 M O+ y! k
6. Unit 22.5 Multi-Color FISH Techniques
" K4 P0 Y% J0 R/ F$ F7. Unit 22.6 Comparative Genomic Hybridization
: S% [) {0 k, b9 ]5 F N8. Unit 22.7 Sister Chromatid Exchange
$ U u9 Z' z2 W U. V8 o7 B9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
1 }- p) o+ f! j$ _% k9 C2 ?% A10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
' ^$ Z5 S) j* z$ U' i+ a$ M' D/ G+ Y11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs7 L' R; M& t% A. O: a
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly4 V) s( F; _( }+ [) r0 K
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells) H6 M, ^' k2 s" S0 x
14. Unit 22.13 Monitoring mRNA Export5 h% G; C' j* P
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-8 B2 @5 f9 Q3 w1 t6 ~
Field Gel Electrophoresis
2 t, P0 M7 X3 D25. Chapter 23 Stem Cells% `/ z# O( c) g/ j$ S
1. Introduction
8 p& ^# b4 s3 D) z, {( J2. Unit 23.1 Stem Cells: An Overview: f q# G, |- _7 i5 k
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
: w5 E/ _0 E2 f g, r0 Y2 I) s Oand Differentiation as Embryoid Bodies
1 S4 t* }7 C" N; O1 S0 T. x4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood5 a+ H, \, A# T
Vessels
- A; i6 Q3 [, z. x5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into2 P% U3 e4 F: h* X
Adipocytes
3 u1 {2 q3 n. x' J! C% f% ^6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
% y; n- C4 h0 p2 g5 J) h7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells- ]+ J" y/ B/ _3 B! O( Y1 h
8. Unit 23.7 Neural Differentiation of Human ES Cells" {0 F! j: m9 N6 X. X4 r/ F
26. Chapter 24 Lipids" c" c, G' I/ y6 g+ Z( z
1. Introduction5 ~9 y: Y! C9 d3 u5 q0 ]5 T8 S" W
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking$ \7 D% U/ O, M" D z Z2 i
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
- v6 H: r6 A& L _" `7 p( j! i4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film: P- P: l/ C; k6 A# E; s
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains6 I1 T9 h: Q& F/ k" F- ?# o
27. Chapter 25 Nanotechnology) y# P* X) m' |* P' U0 Q6 a* L
1. Introduction
: J, D% S, l) c. J% f D5 _2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes/ i t1 W" V! x5 E: _* D
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering$ [1 ?9 }' r6 U
28. Chapter 26 Viruses
1 K9 W* h2 B8 J3 o1. Introduction$ x5 Y0 m1 W! P7 q9 k
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
3 E# {4 R- s* T6 f( m5 Y- h8 r; p. Y3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of6 a0 ` f5 V; ?7 p
Cell Entry
( i" ~! Z) V: M0 G3 U" J" g4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
# w1 D+ g6 K' ]1 S6 e- [1 Y; @5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
* @+ S: s( ]1 d* Y* j' `3 Z4 a6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
`0 f$ U2 k; ]+ S29. Chapter 26 Lipids( u) y+ i6 }4 }' W6 \
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
9 c/ L; A, D6 d0 d: h- x1 Q( ?; DReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication# X1 J7 n7 h2 n8 n0 L; s! o4 Y
30. Chapter 27 RNA-Based Methods in Cell Biology* D1 X3 ]9 Q. I% I( M
1. Introduction
: w( c0 E0 d% C# q+ a$ \$ \8 ]2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
( r5 }' ~) A E$ l3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible. L0 n& H7 Z" n! q: m
Rescue in Mammalian Cells
u: i& w, y8 ?5 B9 y3 k31. Appendix 1 Useful Information and Data# t7 F& ?1 ^$ o8 U. |% B! @$ d
1. 1A Useful Measurements and Data3 P, D1 r/ c8 k2 a2 N
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research
5 k' x0 C! T# G9 _& C3. 1C Identification of Motifs in Protein Sequences
8 e# P/ q# Y4 A5 o4. 1D Safe Use of Radioisotopes% ? D) ^9 `; B1 d! n" G9 D) K* k8 u8 b
5. 1E Absorption and Emission Maxima for Common Fluorophores* F) L: R- H( z: E) v# I) m
6. 1F Importing Biological Materials3 x) O. @$ A, a: U3 _
7. 1G Centrifuges and Rotors |5 Y# ]1 s ]' K% G% D
8. 1H Internet Basics for Biologists
# D0 x! ]8 h4 u3 g U: r6 u5 H7 I' C32. Appendix 2 Laboratory Stock Solutions and Equipment/ o, R/ l: v# [( f/ e$ c# k
1. 2A Common Stock Solutions, Buffers, and Media
) a0 q% G" J' B+ i8 y2. 2B Medium Formulations2 C6 U4 Y' @$ U* f; k9 [ T
3. 2C Standard Laboratory Equipment
7 l* ]# c1 o2 S33. Appendix 3 Commonly Used Techniques2 e( h) j% {- D& M" s
1. 3A Molecular Biology Techniques
! u/ O; y6 c$ q( Z) r! y2. 3B Spectrophotometric Determination of Protein Concentration8 D; ?& J G, V9 P1 h. ?
3. 3C Dialysis and Concentration of Protein Solutions; Y- S, [3 J+ F
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
! b% Q% q: N& D& v8 ]* [5. 3E Silanizing Glassware, F6 }) F1 d% a6 a
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
) x- c* W6 S1 t3 B# h! c7. 3G Micro RT-PCR
" J0 E ~5 F% N4 z1 }- ?: l7 h8. 3H The Colorimetric Detection and Quantitation of Total Protein
4 M* @6 V/ v. S {34. Appendix Suppliers' ?$ q0 Q/ N4 C; c9 F" f) s2 L
1. Selected Suppliers of Reagents and Equipment i0 y' R! V3 W
, T, x# W! b. @5 R6 `2 \3 |
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发表于 2011-3-8 15:53
