Flow Cytometry for Research Scientists: Principles and Applications Chapter Abstracts
Chapter 1
Introduction to the Field of Cytometry and its Importance in Biomedicine
Flow cytometry is a methodology for determining and quantitating cellular features, organelles or cell structural components primarily by both optical and electronic means. Although it measures one cell at a time, the newest equipment is able to process up to several hundred thousand cells in a few seconds. Flow cytometry can be used to count and even distinguish cells of different types in a mixture by quantitating their structural features. Therefore, flow cytometry has great advantages compared to traditional microscopy since it permits the analysis of a greater number of cells in a fraction of the time. In addition cell sorting with flow cytometers has been a powerful tool for diverse fields in biomedical research and clinical applications. This chapter reviews the origins of flow cytometry, describes how it works, and evaluates it importance.
Chapter 2
Overview of Flow Cytometry Instruments
Here the author reviews and compares the various makes and models of flow cytometer available today. The specifications and characteristics of each instrument are clearly described. Both commercially available instruments and home-built instruments are included.
Chapter 3
Flow Cytometric Data Analysis. CellQuest Software
This chapter sequentially describes how flow cytometric data is analyzed and presented graphically in the form of contour plots, dot plots and histograms. Moreover, this chapter about flow cytometric data analysis shows some features of CellQuest software. CellQuest is the software developed by BD with capabilities for acquiring and analyzing samples. It is designed in a user friendly way and permits the performance of off-line analysis.
Chapter 4
DNA Analysis. DNA Measurement and Cell Cycle Analysis
Two of the most popular flow cytometric applications are the measurement of cellular DNA content and the analysis of the cell cycle. Therefore, diverse protocols for DNA measurement have been developed including Bivariate cytokeratin/DNA analysis, Bivariate BrdU/DNA analysis, and multiparameter flow cytometry measurement of cellular DNA content. These analyses have been paralleled with the development of commercial software for cell cycle analysis.
Chapter 5
Molecular Cytometry
Molecular cytometry is a relatively new and versatile technique for addressing cellular and molecular biology questions. It achieves this by combining a number of molecular biology techniques in order to obtain quantitative molecular measurements of single cells. In addition, it permits the analysis of cell-to-cell variations in the molecular parameters being studied.
Some Molecular Cytometry techniques frequently used in flow cytometry include:
· Flow cytometry/cell sorting for cell micromanipulation.
· FISH (fluorescence in situ hybridization) in cytometry.
· PCR (polymerase chain reaction) and TaqMan.
· Image/Confocal Microscopy.
Chapter 6
Surface Staining and Immunophenotyping Using Multicolor Analysis
Most benchtop analyzers are equipped with multiple detectors for performing multicolor analysis. Specimens can be stained with one, two, three and, with some instruments, four fluorochromes to perform multi-color analysis with a single tube. The larger and more expensive cell sorters can routinely perform five color analyses with the possibility of adding additional detectors. For specimens of small sample volume, multicolor analysis offers the researcher the possibility of detecting multiple cell surface markers at one time. This chapter describes the use of flow cytometry in various typesof surface analysis and immunophenotyping, including the immunophenotyping of leukemia and lymphoma samples, reticulocyte analysis, platelet associated immunoglobuling, CD4 T cells, CD34 stem cells, etc
Chapter 7
Handling of Samples: Biosafety
The International Society for Analytical Cytology (ISAC) has created a committee that issues recommendations for biosafety in cytometry and image analysis procedures. This chapter summarises the most important safety precautions required during sample processing and analysis.
Chapter 8
Intracellular Antigens. Cytokines and the Study of Viral Antigens
Cytokines are proteins that play a critical role in the immune system. However, most of the detection systems for cytokine measurements use ELISA methods. Recently, it has been feasible to measure cytokines intracellularly by treatment of the cells with drugs that block the golgi and allow the cytokine accumulation making feasible detection by flow cytometric approaches. Diverse protocols have been developed for identification of intracellular cytokines.
Chapter 9
Flow Cytometric Assessment of Sperm Quality and Cell Cycle Analysis
In order to determine sperm quality, dual fluorescent staining of mammalian sperm coupled to a flow cytometric measurement has been developed. This chapter describes flow cytometric estimation by staining the spermatozoa with two fluorochroms stains, one was the membrane-permeating substrate carboxyfluorescein diacetate (CFDA) and the second was the relatively membrane-impermeant nuclear stain propidium iodide (PI).
Chapter 10
Flow Cytometric Assessment of Allopurinol Susceptibility in Leishmania infantum Promastigote
Leishmaniasis is a major tropical and subtropical parasitic disease. The yearly prevalence is estimated at 12 million people world-wide and 200-350 million people are at risk. This chapter describes a flow cytometric approach to study the effect of treatment methods.
Chapter 11
Flow Cytometric Assessment of Transduction Efficiency and Vector Cytotoxicity of HSV-1 Amplicon Vectors
Herpes simplex virus type 1 (HSV-1) has many properties that make it a promising gene transfer vehicle: (i) the genome is a linear, double-stranded DNA of ~152 kb (1), (ii) at least 30 kb of foreign DNA can be inserted, (iii) HSV-1 can infect most cell types, both dividing and non-dividing. This chapter describes the use of flow cytometry in this area of research.
Chapter 12
Immunophenotyping of DC by Flow Cytometry and Description of Diverse Functional Studies
The characterization of surface markers on human DC has been a very difficult and elusive task because of the lack of appropriate reagents with high specificity for DC identification. However, some molecules whose genes have been cloned and sequenced recently (e.g. CD83, DEC-205) have been found to be strongly associated with DC. Also, a panel of monoclonal antibodies (e.g. CMRF-44) that recognize molecules on DC has been raised. Therefore, there is a growing need to establish a common and comprehensive nomenclature for such known molecules and for the new monoclonal antibodies, as well as to clarify and define the lineage(s) of DC and the existence of DC subsets. Thus, a flow cytometry approach was instigated in order to evaluate the diverse mAb submitted against two DC populations.
Chapter 13
Summary
Flow cytometry is a methodology for determining and quantitating cellular features, organelles or cell structural components primarily by both optical and electronic means. Although it measures one cell at each time, the newest instrumentation is capable of processing hundreds of thousands of cells in a few seconds. Flow cytometry can be used to count and even distinguish cells of different types in a mixture by quantitating their structural features. Therefore, flow cytometry has great advantages compared to traditional microscopy since it permits the analysis of a greater number of cells in a fraction of the time. In addition cell sorting with flow cytometers has been a powerful tool for diverse fields in research and clinical applications.
Chapter 14
Protocols
This section includes protocols for cytometry technics kindly provided by the Salk Flow Cytometry home page and from David Chambers (for coupling and staining procedures) reproduced here with permission of D. Chambers.
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