Cell Separation A Practical Approach

by Fisher, Derek; Francis, Gillian E.; Rickwood, David

• ISBN13: 9780199635801
• ISBN10: 0199635803
• Format: Hardcover
• Copyright: 1999-02-25
• Publisher: Oxford University Press

Summary

Techniques for separating cells are needed in many areas of cell biology. This book presents modern methods from the laboratories of experts in the field, and includes tested, reproducible protocols, hints and tips for success, and troubleshooting suggestions. It will be invaluable to a widerange of cell biologists.

Table of Contents

List of Contributors	xiv	(4)
Abbreviations	xviii
1. Preparation of single cell suspensions	1	(42)
K. M. Cheetham
N. Shukla
B.J. Fuller
1. Introduction	1	(1)
2. Methods of dispersal	1	(21)
Cell isolation from suspension	3	(7)
Isolation of cells from solid tissues	10	(12)
3. Viability measurements	22	(7)
Viability counting with trypan blue	22	(1)
Viability counting by fluorescent dye uptake	23	(1)
Assessment of metabolic functions: protein synthesis	24	(2)
Assessment of cell growth as a viability index	26	(3)
4. Separation of viable and non-viable cells	29	(1)
5. Cell characterization	30	(10)
Light microscopy	31	(1)
Phase-contrast microscopy	31	(1)
Electron microscopy	31	(2)
Flow cytometry	33	(1)
Magnetic beads	33	(1)
Immunohistochemistry	34	(5)
Metabolic characterization of cells	39	(1)
References	40	(3)
2. Fractionation of cells by sedimentation methods	43	(48)
D. Patel
T. C. Ford
D. Rickwood
1. Introduction	43	(1)
2. The theory of sedimentation	44	(1)
3. Separation media	45	(3)
Criteria for cell separation media	45	(1)
Colloidal silica media	46	(1)
Iodinated gradient media	47	(1)
4. Solutions for iso-osmotic gradients	48	(7)
Percoll gradients	48	(2)
Non-ionic iodinated media	50	(2)
Other types of gradient media	52	(3)
5. Preparation of iso-osmotic gradients	55	(3)
Preparation of discontinuous iso-osmotic gradients	55	(1)
Preparation of continuous iso-osmotic gradients	55	(3)
6. Choice of separation method	58	(1)
7. Separation of cells on the basis of size	58	(11)
Differential pelleting	58	(1)
Velocity sedimentation	59	(10)
8. Separation of cells on the basis of density	69	(19)
The separation and purification of blood cells using density barrier methods	70	(2)
Mononuclear cells	72	(2)
Polymorphonuclear cells	74	(2)
The purification of viable spermatozoa from bovine semen	76	(1)
Separation of viable and non-viable cells from disaggregated tissues and lavages of body cavities	77	(2)
The fractionation of cells from perfused, disaggregated rat liver	79	(3)
The isolation of protoplasts from digested plant tissue on OptiPrep gradients	82	(3)
Enhanced isopycnic separation of cells by density perturbation	85	(3)
9. Conclusions	88	(1)
Acknowledgements	88	(1)
References	88	(3)
3. Centrifugal elutriation	91	(40)
Joanne C. Wilton
Alastair J. Strain
1. Introduction	91	(1)
2. Principles	92	(2)
3. Equipment	94	(5)
Separation chamber and rotor head	94	(2)
Centrifuge	96	(1)
Pump	97	(2)
4. The elutriation procedure	99	(4)
Setting-up the system	99	(1)
Elutriation buffer	100	(1)
Number of cells loaded	102	(1)
Temperature	102	(1)
Loading suspension	102	(1)
Cleaning	102	(1)
5. Applications	103	(11)
Liver cell populations	103	(5)
Cell synchrony	108	(2)
Blood cells	110	(4)
Other cell types	114	(1)
6. Advantages and disadvantages	114	(1)
7. Conclusions	115	(4)
8. Simulation of centrifugal elutriation for the Beckman JE-6B chamber	119	(8)
S. P. Spragg
D. Rickwood
Introduction	119	(1)
Basic differential and integral equations	120	(2)
Computation	122	(3)
Correction for wall collisions	125	(2)
Comparison of theoretical with experimental results	127	(1)
Conclusions	127	(1)
Acknowledgements	127	(1)
References	127	(4)
4. Separation and fractionation of cells by partitioning in aqueous two-phase systems	131	(38)
D. Fisher
1. Introduction	131	(1)
2. Theoretical background and its impact on methodology	132	(6)
The influence of polymer concentration: phase separation and phase diagrams	132	(3)
The influence of ionic composition: charge-sensitive and non-charge-sensitive cell partitioning	135	(1)
The influence of polymer ligands: affinity cell partitioning	135	(2)
How cell partitioning arises and the practical consequences	137	(1)
3. Practical aspects of phase partitioning	138	(12)
Selection of phase systems	138	(1)
Preparation of phase systems	139	(2)
Preparation of cells	141	(1)
Single tube partitioning	142	(2)
Countercurrent distribution	144	(6)
4. Selected applications	150	(8)
Immunoaffinity cell extractions	150	(3)
Fractionations of cells into subpopulations	153	(5)
5. Conclusions	158	(1)
6. Manually operated countercurrent distribution apparatus	159	(4)
I. A. Sutherland
P. Eggleton
D. Fisher
Introduction	159	(1)
Description of the apparatus	160	(2)
Applications	162	(1)
References	163	(6)
5. Separation of cells by flow cytometry	169	(22)
M. G. Ormerod
1. Introduction	169	(1)
2. The basic instrument	170	(4)
Introductory comments	170	(1)
Light source	170	(1)
The flow chamber	171	(1)
Optics	172	(1)
Signal detection and processing	173	(1)
Data analysis and gating	174	(1)
3. Cell sorting by droplet deflection	174	(14)
Introductory comments	174	(3)
The principles of flow sorting	177	(3)
Practical considerations	180	(8)
4. Other flow sorters	188	(1)
References	188	(3)
6. Immunomethods: magnetic, column, and panning techniques	191	(22)
P. A. Dyer
P. Brown
R. Edward
1. Introduction	191	(2)
2. Column and panning methods	193	(2)
3. Reagents for immunoseparation methods	195	(1)
Antibodies	195	(1)
Immunomagnetic beads	195	(1)
4. General cell separation using immunomagnetic beads	196	(9)
Coating immunomagnetic beads with antibodies	196	(3)
Immunomagnetic separation strategies	199	(6)
Human peripheral blood cells isolated using Dynabeads	205	(1)
5. Functional approaches	205	(6)
Cytotoxicity	205	(2)
Cell phenotyping	207	(4)
References	211	(2)
7. Separation of cells using free flow electrophoresis	213	(40)
P. Eggleton
1. Introduction	213	(5)
Factors governing the electrophoretic mobility of cells during electrophoresis	213	(1)
The main types of free flow electrophoresis equipment	214	(3)
2. Buffers	217	(4)
Separation chamber buffers	218	(1)
Electrode chamber buffers	219	(2)
3. Preparation of cell samples for free flow electrophoresis	221	(4)
Types of cells separated by FFE	221	(1)
Purification and preparation of cell samples before FFE	222	(3)
4. Preparation and application of free flow electrophoresis for separation of whole cells	225	(13)
Electrode chamber	225	(2)
Separation chamber	227	(3)
Preparation of sample syringe	230	(1)
Running samples in FFE	231	(3)
Construction of cell fractionation profiles	234	(1)
Electrophoretic mobility of cells	235	(3)
5. Evaluation of the electrokinetic properties of cells separated by FFE	238	(8)
Ability of FFE to separate cells with small differences in electrophoretic mobility	238	(3)
Removal of sialic acid from cell surface and effects on cell separation	241	(1)
Application of FFE to study the dynamic changes in the electrokinetic status of cells	241	(2)
Application of FFE to monitor haematological disorders	243	(3)
Application of FFE to study biochemical differences in cell subpopulations	246	(1)
6. Overcoming problems	246	(3)
Cell aggregates	246	(1)
Leaks	247	(1)
Bacterial contamination	248	(1)
Fluctuations of temperature	248	(1)
Fluctuations in separation buffer pH and conductivity	248	(1)
Maintenance of filter membranes	249	(1)
7. Combination of FFE with other techniques	249	(2)
Free flow magnetophoresis	249	(2)
Antigen-specific electrophoresis	251	(1)
References	251	(2)
Appendix	253	(6)
Index	259