Huw S. Kruger Gray (1) and Michael G. Ormerod (2).

(1) Purdue University Cytometry Laboratories,
1515, Hansen Hall, B050,
West Lafayette, Indiana, 47907-1515, U.S.A.
tel: +1-765-494-0757.
fax: +1-765-494-0517.
E-mail: Zip@flowcyt.cyto.purdue.edu
(2) 43, Wray Park Road,
RH2 0DE,
E-mail: 100537.2462@compuserve.com

Summary and acknowledgement.

Data are presented which provide a few simple examples of apoptosis analysis using flow cytometry. With the kind permission of the author, the data files have been taken from the excellent book (available on CD-ROM) entitled Data analysis in flow cytometry - a dynamic approach by Dr. Michael G. Ormerod, distributed by Phoenix Flow Systems. The images show data files analysed appropriately, using the WinList (Verity Software) package, to present examples of apoptosis analysis using total DNA staining as well as using DNA enzymatic in situ end labelling of normal and of apoptotic cell populations.


Examples showing analysis of a "sub-G1 peak" in the DNA histogram.

These data were acquired using a Coulter Elite flow cytometer (Coulter Corporation) and show cells from a murine haemopoietic cell line, BAF3, after fixation with 70% ethanol and staining with propidium iodide (PI). The DNA fluorescence data are displayed as histograms of PI fluorescence, after pulse-processor gating to eliminate cell clumps. During apoptosis DNA becomes fragmented by endonucleases and these small DNA fragments can leak out from the cells, resulting in a reduced total DNA content and hence a "sub-G1 fluorescence peak" from the apoptotic cells.

Figure 1: This shows the control BAF3 cell line, grown with the growth factor IL-3 added and only around 3% of the cells are apoptotic (Ap.). Figure 2: This shows the BAF3 cell line, after IL-3 was omitted for 16hr. and around 47% of the cells are apoptotic now (D = debris).

Examples showing enzymatic in situ end labelling of DNA strand breaks.

In this technique, the large numbers of broken ends on the short DNA strands, resulting from fragmentation of the chromatin at the sites of nucleosomal linkage, are labelled enzymatically in situ usually by means of a terminal deoxynucleotidyl transferase (TdT). Labelling can be directly using fluorescein-deoxyuridine triphosphate (dUTP), or indirectly (more sensitive) using biotin-dUTP followed by fluorescein-streptavidin, or alternatively using digoxygenin-dUTP followed by fluorescein-anti-digoxygenin. Paraformaldehyde fixation then cross-links the DNA into the cells and prevents these small fragments from being extracted. Finally, counter-staining with PI enables the cell cycle phase, from which the cells entered apoptosis, to be observed and gating from a pulse-processor allows doublet-discrimination.

Figure 3: This shows the control BAF3 cell line, incubated with IL-3 and stained using the TdT / biotin-dUTP / streptavidin-FITC / PI method. Most of the cells are visible in the normal cell cycle and few in apoptosis (Ap.). Figure 4: This shows BAF3 cells, incubated in the absence of IL-3 for 16hr. and reveals that many more of the cells have entered aopoptosis, predominantly from the G0/G1 phase of the cell cycle.


Back to apoptosis.

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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