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Data Analysis in Flow Cytometry
a Dynamic Approach
Michael G. Ormerod
©
© © © © © © © © © © © © © © © © ©This book (on CD-ROM) takes an entirely novel approach as it guides you through the different applications of flow cytometry to mammalian biology. Rather than presenting you with text illustrated with static pictures, the book supplies files of the raw data so that you can experiment dynamically with the data for yourself.
In flow cytometry, measurements of up to seven parameters are made on individual cells. Typically, from a single sample, data may be recorded on 10,000 or more cells. These data (known as listed data) are written to the hard disc of the computer. They can be run through the analysis program as often as the experimenter wishes. On each run, different methods of analysis can be tested; it is as if identical cells were being rerun each time through the flow cytometer. On the CD-ROM, 173 of these files have been supplied for you to analyse. They come from seventeen laboratories in seven different countries.
The accompanying text describes the major features of the analysis of data from each application. Guidance is given about how to proceed. You are then free to experiment with the analysis for yourself. Some of the pitfalls are also described and for a few applications, files of misrecorded data have been included to illustrate these pitfalls.
I use WINMDI (supplied with the CD-ROM) for data analysis, but you can use any program which reads FCS data files.
To give you a flavour of the book, I have included Chapter 7 on the quantification of cell death (including apoptosis) together with the first seven data files from the section on apoptosis.
The complete book can be obtained from Phoenix Flow Systems or from the author:
Michael G. Ormerod
Contents
1. Introduction
1.1 About this book
1.2 To use this book
1.3 Key references
2. Elements of Data Analysis
2.1 Introductory remarks
2.2 Gating
2.3 Optics
2.4 Electronics
2.5 References
3. Some clinical applications (excluding DNA)
3.1 Immunophenotyping
3.2 Analysis of CD34
3.3 Detection of intra-cellular cytokines
3.4 Reticulocyte analysis
4. Analysis of DNA
4.1 Introduction
4.2 Pulse shape analysis
4.3 Observing the effects of drug treatment
4.4 Analysis of clinical samples
4.5 Staining viable cells
4.6 References
5. Measurement of nuclear antigens associated with proliferation
5.1 Proliferating cell nuclear antigen (PCNA)
5.2 PCNA - a seven parameter analysis
5.3 Ki-67
5.4 Ki-S1
5.5 Cyclins
5.6 Cyclin B1 and p105
6. Study of cell proliferation
6.1 The BrdU/anti-BrdU method
6.2. BrdU-Hoechst/PI method for measuring cell cycle kinetics in vitro
7. Quantification of cell death (including apoptosis)
7.1 Measurement of viable cells
7.2 Quantification of apoptotic cells
8. Drug resistance in tumours
8.1 Introduction
8.2 Drug uptake
8.3 Intracellular glutathione
9. Functional assays of cells
9.1 Intracellular calcium ions
9.2 Intracellular pH
9.3 Phagocytosis and oxidative burst
10. Miscellany
10.1 Analysis of RNA synthesis
10.2 Combined analysis of DNA, RNA and an antigen
10.3 Chromosome analysis
10.4 Kinetic analysis of intracellular enzymes
Appendix. List of contributors.
Index
Important copyright notice
The copyright of the text in this book is owned by the author, M. G. Ormerod. No part of the text may be reproduced without permission of the author. Figures for use in any publication may not be prepared from the data files supplied without the written permission of the originator of the data (identified in the text). Where the originator is not specifically named, the data were obtained by the author, from whom permission must be obtained.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu