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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑
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Current Protocols in Cell Biology 2010年完整版 5483页
( J- T4 D- }: ~# b5 U* I4 a
% o8 |/ y2 ^; {Online ISBN: 9780471143031
8 E0 X7 s/ U% U- VDOI: 10.1002/0471143030* s9 v; Y0 Q+ e2 \+ m0 d( U" j t5 V
2 t8 f: R7 X: b7 z7 g* Z% Q/ \) N eTable of Contents
! L$ Q a5 V8 Q& w: d7 O- ]- s1. Preface
1 y- J7 n4 i: s$ x o2. Foreword
9 L& ^( P. ?5 N! M" g5 L' B3. Chapter 1 Cell Culture
: [9 h1 n1 [' _1 c P1. Introduction
' @4 C0 E7 X/ j9 C& j/ R+ L) c2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
* P: H1 @9 l, E9 e+ O2 p3. Unit 1.2 Media for Culture of Mammalian Cells
- `/ }- s/ T+ Z7 D4. Unit 1.3 Aseptic Technique for Cell Culture
- z ]7 L7 k3 l+ E# W+ v0 ^5. Unit 1.4 Sterilization and Filtration: H0 E: K; {3 O
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures; U4 i: B3 t! h" }+ Y6 [
7. Unit 1.6 Media and Culture of Yeast
* K A( y0 K5 V, w9 B8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging% r: K C& f1 C7 |# n' t" r
4. Chapter 2 Preparation and Isolation of Cells# M% }5 f$ k+ E: e6 C
1. Introduction
) P( R1 i# G5 |3 f0 |2. Unit 2.1 Establishment of Fibroblast Cultures
t. S6 P) q( @# z1 N5 Q3. Unit 2.2 Preparation and Culture of Human Lymphocytes
- v5 ?- N3 Z2 m' `4. Unit 2.3 Preparation of Endothelial Cells2 R: G" V! h4 S: M. U
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
+ O5 z1 `. i' J7 y! L- B6. Unit 2.5 Laser Capture Microdissection( o7 w: p* q+ e8 a" A# Q
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures* w- Z1 e6 n0 c. h) d7 m
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells2 Z( h y# f3 R% N5 u6 {$ p
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
$ B/ |+ z9 ^. T2 F3 v, A% D Q1 u1. Introduction
- P& V% u: U \5 W8 I n8 K2. Introduction6 D) M8 Z. F2 a
3. Unit 3.1 Overview of Cell Fractionation" m$ v, a7 x* \7 O
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains- U) i3 \8 E. j: O; J0 r2 \3 n' L
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation7 J9 c" n) x3 y& O8 _& m
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
. ]2 B" s9 B4 G2 P+ j. O4 P$ D7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient8 R" @9 ^! o r! T, X
Centrifugation
1 z E k+ h( c1 [4 t, a& W8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient' a8 C- M; o5 n: n
Centrifugation
2 X* j4 O0 a9 l( S1 ?$ h* t9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
8 @# r' A* @7 J2 ~10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
$ S% v9 S3 t7 V! D* b) @11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
7 b* x& A7 w* e- Z6 t, M( m9 c: ACentrifugation: W. V7 b* _1 v
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues* g( G+ Y: A' a8 K A/ [2 T
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
$ u/ ]! q& e: \* D1 A8 a1 D6 Z14. Unit 3.12 Isolation of Synaptic Vesicles. q- [: l( u7 R N1 L
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation9 _* l) t/ @/ r* u
16. Unit 3.14 Isolation of Melanosomes
" [* W9 m# ^" d: I17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation* J: h- J9 o w5 C
18. Unit 3.16 Isolation of Mast Cell Granules' o3 p% M+ \/ r- k0 g2 \& V+ y' g
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
7 v! X& B) [5 J7 J. V0 g20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
& t1 s! m# n& m& S* ?* a21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons4 N5 ^4 G+ J- p1 u# ?( P% [
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
9 T0 }6 j! W! Z) V* ^23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
: S$ Z) ^+ r3 G& Z. D2 ], C% C" s24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
' m, e3 K7 w) u9 b0 U" z+ @Fluids
/ Q2 v4 i- I! d( _4 z' B+ A6 Y25. Unit 3.23 Isolation of Intermediate Filaments3 O% _8 O" J; x5 \ e
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle8 G1 O, |+ }) l
27. Unit 3.25 Isolation of Myelin
' M* p! D, |! z* m28. Unit 3.26 Isolation of Renal Brush Borders$ m: e* C% @7 c9 x
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane- E6 ^$ A W2 T& l1 u
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
' I \' S0 A$ F9 q( o30. Unit 3.28 Isolation of Amyloplasts
4 [) t1 ?& n5 S, Y* R8 W- s31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins" _$ }9 F/ @4 V- T, R6 r4 m
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic& ~" {, y, o- e# l
Centrifugation4 ]) g( Q$ U( }" S1 h1 g0 g6 y: h
33. Unit 3.31 Isolation of Neuromelanin Granules
7 i6 Y7 |- j- K7 t8 `) e34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
3 ~# a/ {3 |6 zDensity Gradient Centrifugation) i% x% t# L$ H
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments j( `) B; a. n
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation5 ?' p% a- R* W" F; ^" v3 b
37. Unit 3.35 Isolation of Platelet Granules' Z3 [" P3 R$ {5 d! L
38. Unit 3.36 Isolation of Nucleoli
- S; l& m# Q+ @/ @+ H, n" B# q8 G39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins4 ^7 x& B' k9 s, h9 q- j) [
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
h& g. @" O4 `9 e5 ?" y V- u41. Unit 3.39 Isolation of Chromaffin Granules1 _1 y8 e3 J! ^3 e+ s$ O) o
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
# N6 t: J$ [( r5 V6. Chapter 4 Microscopy
. E% W& w$ W' l1. Introduction
! B/ D/ H# a9 G+ H; g2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope3 H* ~; A( R2 {$ G0 Y. C
3. Unit 4.2 Fluorescence Microscopy
# O5 Z8 F( v- i4. Unit 4.3 Immunofluorescence Staining" t/ |0 A1 [8 U- l/ a
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria i+ N6 W4 @$ A3 Y
6. Unit 4.5 Basic Confocal Microscopy, e) ]* B* U4 ^4 I% [4 @% w w
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
7 U' a% [" q1 q4 v, ?- A. w8. Unit 4.7 Cryo-Immunogold Electron Microscopy
' y& ]+ o1 c- T9 H5 O9. Unit 4.8 Correlative Video Light/Electron Microscopy; O+ D& Q3 B& d& D h
10. Unit 4.9 Polarization Microscopy( l+ Q. {+ M. V4 R: F9 K$ T. }
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells7 S( K* ^1 ?/ v$ ^+ w4 q; H `
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues; B: \: o1 _+ l' B2 p5 m
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface2 v5 l, [) ^3 g0 r
Events8 f6 C- i* ^# J* U
14. Unit 4.13 Fluorescent Labeling of Yeast
, K. U5 q; P9 e2 j: I9 w5 ]3 p9 w% x15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
& W5 D/ p* j. g7 ]" [16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy% \7 U7 x0 D, f
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules3 e1 y0 k5 z3 ]5 P" y
in Endocytosis
7 V& \. p1 A+ `% n6 d18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
7 _" g1 D) Q# r. ~ c. m19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development$ u$ z \) S9 W$ g; S
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
9 A) h! `' I- I21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
' Y8 S2 J9 |6 R+ P' u22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
" V; q9 v) A$ F1 O9 e23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
( n* C5 c/ r4 P8 T" @8 @24. Unit 4.23 Interference Reflection Microscopy
' l; J! W |7 Z25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach& n' a. @: \+ r4 v* y5 A" y; w
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
. p6 {: Z* u ]& o27. Unit 4A Organelle Atlas: Appendix to Chapter 4
+ b1 F/ }3 I' m! d- R* ?4 n2 o7. Chapter 5 Characterization of Cellular Proteins& F( U" }7 M) }) y
1. Introduction( o$ n8 ?' n3 B9 e9 F Q& k. G
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells. F1 g( J. [) g c& c
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein" Y( f( Z" n- B$ b. H6 N2 D
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
8 N) a8 Y$ V K7 G6 [5. Unit 5.4 Analysis of the Association of Proteins with Membranes
8 y( ]' E- a/ }7 f" R' y @( ^6 ^6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
8 t$ ]8 e9 h8 a7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
7 F) _4 w5 [9 C+ S& H0 fSpectrometry
/ U4 R0 V2 n' Z4 [ u3 Q3 W8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
$ I: L( X- @* n& IApplications
8 _: O% f3 W6 y/ r M7 u6 y9 ^; d8. Chapter 6 Electrophoresis and Immunoblotting
0 ?$ x. r% r2 Z2 o7 M& B; f1. Introduction. u+ x; s* J7 B+ S' [) K/ T7 s
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
1 @ g" H# `2 T) `3. Unit 6.2 Immunoblotting and Immunodetection
) W% l6 y. \: I) r9 S) A4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots: i, v$ [& a/ @: g) J* R
5. Unit 6.4 Two-Dimensional Gel Electrophoresis5 M' I& ~* r7 e9 z* S
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
, o3 O6 E3 }( n, i- ~: w6 W/ G7. Unit 6.6 Staining Proteins in Gels
& V6 J( `8 K! w! o8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
6 {7 o" u3 K+ g. B/ G& ~: Q9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots& K5 h( P! |8 ]6 a
10. Unit 6.9 Digital Electrophoresis Analysis
7 W2 d4 ?! Q! O9 H+ z8 b$ ~ G11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis; f7 F v6 S- z: v3 @+ g
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of& h% y% k) d) t6 z" K9 P& ?$ {6 i# Q
Single Cell Gel Electrophoresis (Comet Assay)
- Q! v% u0 V% h9. Chapter 7 Protein Labeling and Immunoprecipitation7 M/ N. h7 O I: U7 U3 N
1. Introduction4 X% f/ y( i) Z& H& {) D
2. Unit 7.1 Metabolic Labeling with Amino Acids9 e) ~2 F3 |' F# b- W
3. Unit 7.2 Immunoprecipitation
) e3 r* E7 K2 a; \4. Unit 7.3 Metabolic Labeling with Sulfate/ j- n5 j1 F h+ r$ O4 E! U
5. Unit 7.4 Metabolic Labeling with Fatty Acids; Z, t3 G9 s2 u0 u' y4 w
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
! G# V) B- z; B7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
5 @" V1 T; C7 t5 p7 G: m8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins, |. `) ~3 r, f! @- N
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars( H5 a* i' I8 w0 ~+ n
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
) h' o- y. o3 B. t j7 A11. Unit 7.10 Radioiodination of Cellular Proteins. s) I. Q' D: t$ `, ~
10. Chapter 8 Cell Cycle Analysis. P( K( ~1 a6 O; F
1. Introduction9 S# ?& c* w, L- _
2. Unit 8.1 Overview of the Cell Cycle
/ i/ l( Z* F8 ]+ R( X2 t3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle: K8 c+ F4 [7 }& e1 \. }$ h
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle& _+ v: w- T5 ]( E$ L
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry5 b' }- T5 b2 ?5 z! b
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
/ b/ M& i7 F* Y3 ~7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
3 T% H- M+ \3 J6 n2 Z/ d0 X4 ~11. Chapter 9 Cell Adhesion
( Z% ?4 W) \1 j) n$ ? @1. Introduction
( J. O0 ]& @4 R$ [ U2. Unit 9.1 Cell-Substrate Adhesion Assays
' }0 T! @7 M$ X2 v3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force2 P' o% E7 i& i! p* T! n6 X
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
d# \& j, k: G1 {5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
6 \: L$ j4 B: N, J+ [ O6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules1 v4 u; B% A# {# z1 M) m
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions$ A1 ~. |1 ?+ r' q- z" m4 z. I& x0 k3 _; ]
12. Chapter 10 Extracellular Matrix9 z5 D7 l5 ? |
1. Introduction) I, v: k8 I) b" T$ W
2. Unit 10.1 Overview of Extracellular Matrix+ ]. S" T( ]7 z2 l1 _3 R
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
0 p9 \ b/ {# X5 _( W! q5 k+ L' c8 ~4. Unit 10.3 Preparation of Gelled Substrates
5 ^2 _: U0 R$ |6 P4 C$ i9 W, K% k5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
( q* g6 z) Q0 {9 d+ K( _0 vEndodermal Cells8 s' x# g3 K! v7 M$ D" S
6. Unit 10.5 Purification of Fibronectin
& U6 i H1 y2 d' |9 l) ]7. Unit 10.6 Purification of Vitronectin( O" _# X, L1 L( q
8. Unit 10.7 Proteoglycan Isolation and Analysis5 n$ o& Q+ {0 x; t# R
9. Unit 10.8 Matrix Metalloproteinases
- ~5 B C# @- P8 z) G3 {10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts o8 W% \, J/ p3 V% H& ^
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
' b0 E0 W0 |, f$ ?: ]" ~# [! z12. Unit 10.11 Purification of SPARC/Osteonectin
* \5 ?7 G: B) ?5 w+ [$ s13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
; D( i# S' J, ~/ o5 R14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
) l8 {- ~: Q; w! Z5 @! J, K6 ]15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor/ [! _) O& X5 q2 k" }
Xenografts
& \- s" G7 R9 E7 z8 q! j7 V$ U16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning* w: N# Q7 h( ~2 q4 R
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties& e: A" A. e6 n4 b, F
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D+ B8 \. h( U& P/ _' B
Cell Migration q+ [ r+ J" n$ q" ^8 [
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
- X- g( o+ z' V1 Z' t$ r L$ C13. Chapter 11 In Vitro Reconstitution& Z# R# `3 O! N! x/ @
1. Introduction
6 j* J- z7 p5 \: m, X2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
9 Y" B! {# q: h& W4 q# L! N- @3. Unit 11.2 In Vitro Translation
% W6 v6 h, f. {% Y4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
# Y3 e5 E+ C7 ~: a3 z* }+ L* S5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
6 ^/ \% d% o5 W' B" v* E6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication" x4 q( {# v: y" C3 n
7. Unit 11.6 In Vitro Transcription; o W" k. r9 s- q
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells: @* y: r' v# m6 |
9. Unit 11.8 In Vitro Translation Using HeLa Extract
4 r3 N. b* X+ G: y5 l$ x: N4 r10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems2 q8 j1 ]) S4 e8 w0 l/ @6 U! U3 [
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts$ N' D7 V% B `5 @
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
0 v0 O5 y: [1 w. v: C13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
6 J4 v6 m6 g" N+ J/ y( i5 Y14. Unit 11.13 Mitotic Spindle Assembly In Vitro5 K& y. t3 n, x+ h5 ~ X
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
3 ^ T5 f2 e- K1 q$ a16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import S; i* U# p* W* T) w7 D
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import" Q& k+ S7 Y7 A4 x( R, {
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
/ b- h; j6 w8 g2 [7 M2 ?19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells. F! s( Y" E0 u: o1 M( k4 T
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import( |% o1 ?9 [9 i' `4 y9 I
14. Chapter 12 Cell Motility$ {7 N! u6 y7 X l" q/ f4 [
1. Introduction$ r2 d1 g7 ^* T. Q! t
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells6 l+ j' r, s& c2 j) ^3 A. h5 [9 L
3. Unit 12.2 Invasion Assays
1 h* b5 r% Q$ S+ F' R8 H [4. Unit 12.3 Cell Traction
+ |8 Y% Q( j5 N( B5. Unit 12.4 Cell Wound Assays
! M( d0 f* m2 b/ R- Z6. Unit 12.5 Dictyostelium Cell Dynamics
" ?" I5 x' _8 N' ]& {; s7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
$ G$ u& f) t, V7 W( F9 j8. Unit 12.7 Actin-Based Motility Assay
! I0 m5 ~* Q$ U" M7 V4 \+ `0 w9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
1 h4 L) Z+ c" {0 e15. Chapter 13 Organelle Motility j; K" U6 Q1 e6 n* L0 v
1. Introduction
+ j; M# }9 ?/ _; w0 H/ V2. Unit 13.1 Microtubule/Organelle Motility Assays: _; J8 R# x0 L1 S' O' Y, a- w
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin2 g8 v# c3 ]6 C& @' ]
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
) R+ E3 ~% @9 c1 O5. Unit 13.4 Movement of Nuclei
@- e: J. p* R9 b" y6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching5 v0 ~* b) b9 ^9 w% R) `
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly0 e; H* G) I; j" z9 |. w3 H) s/ c
16. Chapter 14 Signal Transduction: Protein Phosphorylation
0 _, }% r" E! B4 d' W+ o( O1. Introduction4 f. ?3 j! j' E" Q% d. k2 l/ W
2. Unit 14.1 Overview of Protein Phosphorylation
# }4 g- _" U5 T0 {$ t4 v3. Unit 14.2 Immunological Detection of Phosphorylation
" s# R+ r9 k# b$ H- D5 x4. Unit 14.3 The Detection of MAPK Signaling
( G* _" ^3 N% O0 P! x4 X2 H5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
x1 |1 @1 o2 s. `7 J/ ~" x6. Unit 14.5 Phosphoamino Acid Analysis2 u; J2 H7 _3 Q0 Q+ H+ v
7. Unit 14.6 Determination of Akt/PKB Signaling
% h K/ z o- ?( ^* V( ~6 H8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events: K4 h; p( r9 o3 B! R H
9. Unit 14.8 Rho GTPase Activation Assays1 q- ^% `: P, } i1 P1 N/ d
10. Unit 14.9 In Vitro GEF and GAP Assays
; q+ r' ^ K: ~; |0 L11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
8 u3 A2 l, z0 Q3 r& U! {) DEnergy Transfer (FRET)
% F3 n7 t4 q: B4 h: P3 \; l12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
0 E7 I, T s$ A- w. E2 b3 _13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells( \' v, k, V1 y; m7 \! r
17. Chapter 15 Protein Trafficking3 f4 q8 j! X( \; E1 ~
1. Introduction
6 f- [( A, H0 c t2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
. N& j' p( n' @! _7 t' ]) e0 g# i/ I k3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
4 j$ e0 H6 \' w& v6 p6 ]6 a# w4. Unit 15.3 Endocytosis: Biochemical Analyses
4 ]0 W* H6 e) T# c1 s, Z9 [+ `5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
$ O3 X& n+ k3 `. E6 X Y6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells2 M' Q1 a# y9 S. O& S8 r; M# S- Z5 c
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum4 r* Z$ Z! R5 f1 A7 [# \# o- Z
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation' R5 g+ c& c" }
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
4 N1 |% ]6 m7 D2 L/ ^1 y1 G10. Unit 15.9 Studies of the Ubiquitin Proteasome System$ F$ |! Y% }) ?7 I$ D! _& |9 R# l
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
8 C! R# ]1 w. n. M* B4 g12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
1 q; o# f! p J13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells7 b0 G2 @5 P) P
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging, F5 \/ I* P0 x8 [& g
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
1 V( {" n6 G! e3 ]+ _& Z2 fEGF Receptor
+ U8 ?6 ?0 b/ J0 c" f, V" G16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers2 T* |: \1 d& ^5 p" t! t5 p
18. Chapter 16 Antibodies as Cell Biological Tools
5 \1 t; D: v! x1 k7 Z6 n# {1. Introduction- T/ Q& H8 \( E1 ^3 {
2. Unit 16.1 Production of Monoclonal Antibodies
! q# a& L* B+ M8 C6 h3. Unit 16.2 Production of Polyclonal Antisera
/ [7 x# r# C: m$ m: Z4. Unit 16.3 Purification of Immunoglobulin G: |0 \' G( B3 |% h
5. Unit 16.4 Fragmentation of Immunoglobulin G
3 {1 M7 s. E- z2 N2 o" G) }, @$ `2 u6. Unit 16.5 Antibody Conjugates for Cell Biology0 x& \8 o/ x) q% Z( f( \, b; h* U+ o
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides; K( O4 g, K O* t7 h
19. Chapter 17 Macromolecular Interactions in Cells9 J0 |7 d3 F- j% J2 x
1. Introduction* v& T$ w7 w% w7 H4 N% d& G
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
# `0 G% n* h' ] j' v! CMicroscopy I% x/ I3 ]3 W- h
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis# o; I; C; d/ f# y( g0 \" M
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
. b a2 y# f6 v# C% W, a/ |5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries/ Z* r0 n- T- V) C! z- I* @. O* ?
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
; S1 `$ ?1 E3 @: `7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
- K7 D5 U9 C7 M7 {4 v3 N8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
! j& F9 v& l v, E+ L! bGenomic Sequences In Vivo
" i( D& R1 X1 [, L; _* a9. Unit 17.8 Isothermal Titration Calorimetry( [/ a$ F1 ?) m. W% ~3 j; X
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
) q; w3 K" |! Y0 X8 ]. o( i$ N7 W+ I11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
) ^, Y' }4 f4 T1 U9 {12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
! V' @. e( H3 J, G" cInteractions (RFAP) in Live Bacterial Cells% ?( o0 U. f! N5 W ^' _# h- Y1 L
20. Chapter 18 Cellular Aging and Death
- @; f7 Q+ [ L! N; t1. Introduction8 N9 G- k0 r/ W0 o
2. Unit 18.1 Current Concepts in Cell Death- X1 l5 w3 F6 n/ H9 X. {& D0 }" Y
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis7 F& }! \ R0 i) @' |+ ~' P: ^. K
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria |0 Q) h- |0 P1 M
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
% L' z3 n& `. y! [6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death! r E0 t8 b7 i
7. Unit 18.6 Analysis of Telomeres and Telomerase% F0 B1 c* n. x+ x+ c# r/ i! H
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of4 B6 n6 n0 D* X
Poly(ADP-Ribose) Polymerase* k9 R: s n0 S2 a) ]2 h' g9 z3 j
9. Unit 18.8 Flow Cytometry of Apoptosis2 O" G, m+ ]( v) Y$ Z$ o1 ?
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase$ y0 b4 `+ Q) L- B M
Assay h8 W' F# a4 y5 z# S' i
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
4 ]+ Z$ Y- {! d) ]* |, N6 L21. Chapter 19 Whole Organism and Tissue Analysis
4 [) |$ ?) D- ]4 B Z7 N$ ~" ?! b1. Introduction
" W9 `8 D# v) B1 o) V4 S2. Unit 19.1 Overview of Metastasis Assays; y: Y- W c; X) s) f1 O
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
% N4 g/ T& v! ^: e2 o, Y5 Y9 H7 T4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
; d6 [ Z# }' b7 m% T5 z1 qExpression After High-Fidelity T7-Based RNA Amplification0 D: [ t6 d% l/ y3 X9 c
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA) [$ y. L# K0 @0 D
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model% M0 W! q$ j' [- ?* z! z3 n ]
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
* A) X% q5 @$ w/ g6 B9 M0 k4 s8. Unit 19.7 Imaging Tumor Cell Movement In Vivo4 e' V# o7 m( Q8 l$ B
9. Unit 19.8 Embryonic Organ Culture
6 V, ]* h9 ]# |! H10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
3 U) w7 u+ z' M) m+ v6 G$ }* n7 o11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice# Z/ V5 B) c. Q4 d
12. Unit 19.11 Generation of Transgenic Mice
) [, V8 M }4 x9 o13. Unit 19.12 Overview: Generation of Gene Knockout Mice
1 O) U5 d N$ J" c/ X7 K6 x0 F- B14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
1 o. v S2 L K9 c15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection M8 k. n/ _( a4 Z. q4 M
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
' |9 Q5 x% P, \" l Q0 H! _1. Introduction
7 q# v, M7 z( O4 \8 Y: K2. Unit 20.1 Direct Introduction of Molecules into Cells. m8 p8 S! b" v
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System. W: V! z4 v+ _7 e& B
4. Unit 20.3 Calcium Phosphate Transfection0 _$ t; q! V$ I' {8 ?3 [
5. Unit 20.4 Transfection Using DEAE-Dextran
# j4 T5 u" O1 Q2 h6. Unit 20.5 Transfection by Electroporation2 l( x, |, p0 @) T
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents# L8 q3 w3 @2 C! Q5 x
8. Unit 20.7 Optimization of Transfection
. O: m Y h' G1 l3 U$ ?9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System# o d0 e4 Q4 M7 O5 c) ]$ }$ {
23. Chapter 21 Fluorescent Protein Technology
. J" |- U# d! j$ P; S5 |# Q1. Introduction
$ K3 L% N2 z5 |6 k2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells Y4 X' t% I$ ?8 _; v
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
& H9 z% v6 j' }8 S& }$ t4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
" l6 z0 j) Z! z: }9 {Complementation (BiFC) Analysis
: G9 [. }( y1 D" }0 {/ f3 s5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology0 y) t0 F" C6 ^) O* v# h
6. Unit 21.5 The Fluorescent Protein Color Palette" f5 s& l4 ` i! O6 T3 x" ?
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins- U$ ]; d. S; ` B
24. Chapter 22 Cell Biology of Chromosomes and Nuclei
% l o2 I) i5 C1. Introduction
_! k' {5 j2 S- j1 h5 c) o0 V ]2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis+ R5 i( q" d8 u0 V C% K. Q+ _
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
3 I! h8 C) R9 d4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis$ d& u( c" {9 D0 q& }; C% U o# I
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
$ J8 Y" c% k0 u7 _ F- W6. Unit 22.5 Multi-Color FISH Techniques
+ F# f: S$ O3 C8 \7. Unit 22.6 Comparative Genomic Hybridization
: f) Q3 M- K* ^. I7 i8. Unit 22.7 Sister Chromatid Exchange1 ?% m3 ^* `( R9 t& _1 h
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery, J2 ^$ z9 J5 @5 G P6 D9 P6 o) t
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
5 I& h' j7 O+ S0 O4 F" j11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs1 l7 K7 e3 f1 ]7 Y( T6 ?4 W
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly. m, v( q8 B: K. `
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells5 ]) |3 R& i1 g$ d; l. m, o% A; d
14. Unit 22.13 Monitoring mRNA Export
3 {& ^/ C" ~7 {$ z6 p$ D15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
8 K3 x4 N2 v; c v# wField Gel Electrophoresis* f- N5 F8 v- f0 o5 N8 A7 d
25. Chapter 23 Stem Cells
- O h- q z" J8 `; ?1. Introduction, k, Z+ _2 K' k7 S' @8 a; Q
2. Unit 23.1 Stem Cells: An Overview
3 L g# I' A, J2 |9 p! S8 K, g3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
5 c4 O+ F8 p1 band Differentiation as Embryoid Bodies
) ]0 ?' A5 C8 j1 w' W/ I' d1 h5 W2 h4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
3 \5 i, C1 Q Z0 B# Z4 DVessels
+ h7 b5 v J- O5 y1 R! h5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into9 Z# R, b) }5 C! ^
Adipocytes5 ^: H! k4 N, o5 r) T% e2 k
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
5 _. L1 [' \* Q; ^0 Z* z1 f7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
: n: C& x' b& V4 ~8. Unit 23.7 Neural Differentiation of Human ES Cells
8 U3 ?" T0 y9 [/ j26. Chapter 24 Lipids
3 ?- o2 r, `. P, l/ m* t1. Introduction
9 v7 r' {# B$ c0 K4 q2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
- H3 @( G x6 ]$ X3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins: G$ y2 J5 Z& x9 B; V; Z
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film# C/ T3 y5 z5 e5 D8 y( @+ q
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains L: c$ p$ P0 X( _
27. Chapter 25 Nanotechnology5 y# _; O* M! Y$ i3 p
1. Introduction
6 g7 v0 G. k0 {& P2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
( g4 S3 D( \) y: Z0 B6 d3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering! \/ V# e6 t$ ~( j8 B9 H
28. Chapter 26 Viruses
5 K5 l/ [; i6 w1 S* w1. Introduction$ x4 \; ~! W! [% h" w7 e* R2 g
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors" Y- b& \5 H1 p$ b% U2 y: [- {% ~
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
. I, A! N u: i, MCell Entry; o+ {' [& e- y6 d$ K$ B4 @1 ]
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection$ l. y" ]3 \& }
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
& ~# k. S$ k& M4 ^( k6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
P$ i$ e; U: O6 q, c29. Chapter 26 Lipids
8 @' t) ^* `; U9 N. ~/ L1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by* _6 }* Y' O' ^7 d7 _( G
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
2 h! Y# [! N) L8 U30. Chapter 27 RNA-Based Methods in Cell Biology
9 }. A1 ]9 `$ Q- T7 C0 ]4 H1. Introduction( a; }6 t) K- h' M4 ^% c9 B
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs. n. |6 g* ?9 ^! a( t8 R
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible v) d6 o8 s/ M: V! v* A
Rescue in Mammalian Cells
( H! v! u; R8 s2 E5 D31. Appendix 1 Useful Information and Data, i+ B9 s: M" Z; U& _
1. 1A Useful Measurements and Data$ a; ~2 n: A; n9 @) a9 d( b
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research. L5 c$ D, O+ l7 T6 z- J$ C
3. 1C Identification of Motifs in Protein Sequences
' c0 I# l& P }9 W, @' k4. 1D Safe Use of Radioisotopes
, I' p& R; x: H% N5. 1E Absorption and Emission Maxima for Common Fluorophores4 F J; s. D4 A5 ]2 Q5 o
6. 1F Importing Biological Materials; U* ]$ ?7 ^9 B; |$ f8 s
7. 1G Centrifuges and Rotors, ?) {; U: z; j3 Z% t
8. 1H Internet Basics for Biologists
$ ~. ?: ?. N5 }. y' w) y2 Y32. Appendix 2 Laboratory Stock Solutions and Equipment
& x) P3 C; k; M) F. ^2 C1 A1. 2A Common Stock Solutions, Buffers, and Media
3 X6 P% S; ]9 U2. 2B Medium Formulations% `! X+ x2 c$ O% _ z7 S
3. 2C Standard Laboratory Equipment
2 R& z% b1 ~$ Y7 d0 F33. Appendix 3 Commonly Used Techniques+ ^# [# Z4 d! m7 y* w6 b( x. f
1. 3A Molecular Biology Techniques
6 s* I% D c: m: G2. 3B Spectrophotometric Determination of Protein Concentration: w8 r( {2 J' S% M9 s
3. 3C Dialysis and Concentration of Protein Solutions
% f3 U& ?8 i1 B' |2 ~- P* Z4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy5 M8 {1 y5 N9 x( s" q3 S
5. 3E Silanizing Glassware
+ E( M, L3 G9 E6 c! \: u N5 b6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization, k8 b4 r9 a! D. I5 x
7. 3G Micro RT-PCR
% ]: E8 g% {, }8 r Z2 E8. 3H The Colorimetric Detection and Quantitation of Total Protein
1 p; s- u& Y& G9 K$ {4 [34. Appendix Suppliers
4 Q' Q) O0 |$ ?/ |1. Selected Suppliers of Reagents and Equipment
f, K! d! g: T& {( V) E
& @) P! p# M0 ?6 ^ |
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