Riassunto
Presently, the need for methods involving separation, identification, and characterization of different kinds of cells is amply realized among immu nologists, hematologists, cell biologists, clinical pathologists, and cancer researchers. Unless cells exhibiting different functions and stages of differ entiation are separated from one another, it will be exceedingly difficult to study some of the molecular mechanisms involved in cell recognition, specialization, interactions, cytotoxicity, and transformation. Clinical diag nosis of diseased states and use of isolated cells for therapeutic (e. g. , immunotherapy) or survival (e. g. , transfusion) purposes are some of the pressing areas where immediate practical benefits can be obtained by applying cell separation techniques. However, the development of such useful methods is still in its infancy. A number of good techniques exist based either on the physical or biological properties of the cells, and these have produced some valuable results. Still others are to be discovered. Therefore, the purpose of this open-end treatise is to acquaint the reader with some of the basic principles, instrumentation, and procedures pres ently in practice at various laboratories around the world and to present some typical applications of each technique to particular biological prob lems. To this end, I was fortunate to obtain the contribution of certain leading scientists in the field of cell separation, people who in their pioneer ing work have struggled with the particular problems involved in separating living cells and in some way have won.
Contenuti
1 Transient Electrophoretic and Sedimentation Analysis of Cells in Density Gradients.- I. Introduction.- II. Instrumentation.- A. General Considerations.- B. The Single-Column Instrument.- C. The TRANS-Analyzer Prototype System.- D. The TRANSANALYZER™.- III. Data Acquisition and Processing.- A. Data Acquisition.- B. Smoothing of Data.- C. Slope Analysis.- D. Peak Detection and Baseline Correction.- E. Moment Analysis.- F. Decision Boundaries.- IV. Methodological Aspects.- A. Physical Description of Cell Transport.- B. Absorbance of Cell Suspensions.- C. Density Gradient Electrophoresis.- D. Density Gradient Isoelectric Focusing.- E. Velocity Sedimentation at Unit Gravity.- F. Cell Samples.- V. Applications to Cell Analysis.- A. Analysis of Lymphocytes.- B. Analysis of Red Blood Cells.- C. Conclusions.- References.- 2 Electrophoretic Light Scattering and Its Application to the Study of Cells.- I. Introduction.- II. Principles of Electrophoretic Light Scattering.- A. Electrophoresis.- B. Laser Doppler Shift Spectroscopy.- C. The Light Scattering Apparatus.- D. The Electrophoretic Light Scattering Chamber.- III. Cell Electrophoretic Light Scattering.- IV. Conclusion.- References.- 3 Cell Separation by Endless Fluid Belt Electrophoresis.- I. The Objective.- II. Rotational Suppression of Thermal Convection.- III. The Circular Endless Belt Apparatus.- IV. The Noncircular Endless Vertical Belt Apparatus.- V. Endless Belt Electrophoresis as a Form of Deviation Electrophoresis.- VI. Properties of the Endless Belt System.- VII. Modes of Operation.- A. Single-Order Collection.- B. Split-Order Collection.- C. Isoelectric Accumulation.- D. Nonisoelectric Accumulation.- E. Zonal Separation.- F. Multiple Separations.- VIII. Thermal Convection Artifacts.- IX. Preparative Instrument with Removable External Magnets.- A. Major Components of the Cell.- B. The Iron Core.- C. The Injector System.- D. Plug-in Windows and Collector.- E. Centrifugal Buffer Flow.- F. Regulation of Buffer Flow.- G. Sample Injection.- H. Protection of the Buffer Delivery Line.- I. Overall View of the Apparatus.- X. Preparative Instrument with Submerged Magnets.- XI. The “Fan” Collector for Increased Resolution.- XII. Operation of the Instrument.- XIII. Measurement of Electrophoretic Mobilities.- XIV. Examples of Electrophoretic Separation of Living Cells by Endless Belt Electrophoresis.- A. Buffers.- B. Yeasts and Bacteria.- C. Mammalian Cells.- XV. Theoretical Considerations.- A. Tangential Buffer Flow.- B. Lateral Buffer Flow.- C. Idealized Particle Motion in the Endless Belt.- D. Impairment of Preparative Resolving Power by Mismatching of Horizontal and Vertical Velocity Profiles.- E. Superposition of Electroosmotic Streaming.- XVI. Resolving Power.- A. Flow Fluctuations.- B. Distortion of Streak Profile.- C. Definition of Resolving Power.- D. Resolution in Collection.- E. Band Rectification by Matching Electrophoretic and Electroosmotic Velocities.- F. On the Possibility of Universal Band Rectification.- References.- 4 Buoyant Density Separation with Linear Gradients of Bovine Serum Albumin and Analysis by Centrifugal Cytology and Flow Techniques.- I. Introduction.- II. Gradient Separation Method.- A. Density Gradient Media.- B. Preparation of BSA and Salt Solutions.- C. The Effect of pH on Buoyant Density.- D. The Gradient System.- E. Hydrodynamic and Wall Artifacts.- F. Data Analysis.- III. Past Procedures for the Preparation of Fixed Stained Cells from Dilute Suspensions.- IV. Centrifugal Cytology.- V. The Isolation of Heteroagglutinates.- A. Introduction.- B. Neutral Density Separations.- C. Control Experiment.- D. Method of Cell Agglutination.- E. Gradient Separation of the Heteroagglutinates.- VI. Combined BSA Gradient Centrifugal Cytology Studies.- VII. The Design and Implementation of a Flow Analyzer for Cell Separation Studies.- A. Fluid System.- VIII. Conclusion.- Appendix A. Fraction Collector Controller Circuit.- Appendix B. Buoyant Density Distribution Program.- References.- 5 Physical Separation and Characterization of Reticulocytes and Other Cell Fractions from Rat Bone Marrow, and the 1g Mini-Staflo.- I. Introduction.- II. The lg Mini-Staflo.- A. Multichannel Variable-Speed Pump.- B. The Flow Cell.- C. The Collection System.- III. Model Experimental Studies and Theory.- A. Sedimentation.- B. Separation Criteria.- C. Theoretical Model Studies.- D. Experimental Particle Separation.- IV. Rat Bone Marrow Cell Separation.- A. Cell Suspensions and Fractionation Conditions.- B. Compositions of Cell Fractions.- C. Cytology of Cell Fractions.- D. Sizing of Cell Fractions.- E. Deformability Measurements.- V. Discussion.- VI. Summary and Conclusions.- References.
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