Example DNA analysis data.

EXAMPLE DATA FILES SHOWING DNA ANALYSIS BY FLOW CYTOMETRY. 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, Reigate, RH2 0DE, England. E-mail: 100537.2462@compuserve.com

Summary and acknowledgment.

Data are presented which provide a few simple examples of DNA 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 pulse-processing to eliminate cell doublets and of normal as well as of aneuploid cell cycle distributions.

Examples showing cell doublet discrimination.

These data were acquired using a Coulter Elite flow Cytometer (Coulter Corporation) and show cells from a murine leukaemia cell line, L1210, after fixation in 70% ethanol and staining for DNA with propidium iodide (PI). In each figure, region R1 has been set on single cells and region R2 on cell clumps. For accurate analysis of cellular DNA content, it is important that only single cells should be analysed from a DNA histogram, since a cell in G2 will have an identical DNA content as two cells (ie. a doublet) in G1 of the cell cycle. Analysis of the relative dimensions of the fluorescence signal pulses permits this, because of the differences between the height, width and area of the respective pulses generated from single and multiple cells in the various cell cycle phases. For instance, a single cell in G2 will display an increased pulse peak height relative to its pulse area, whereas a cell doublet in G1 will display an increased pulse width with respect to its pulse area. Consult the literature for extensive and detailed explanations of doublet-discrimination using pulse-processing.

Figure 1: This shows the L1210 cell line, with fluorescence pulse-processing as a cytogram of pulse peak (height) vs. area (integral), to resolve single cells from cell doublets and clumps.

Figure 2: This shows the L1210 cell line, with fluorescence pulse-processing instead as a cytogram of pulse width (time of flight) vs. area (integral), to resolve single cells from cell doublets and clumps.

Examples showing determination of DNA content of normal and of aneuploid cells.

Figure 3: This shows the L1210 cell line, after fluorescence pulse-processing to eliminate cell clumps, as a histogram of propidium iodide (PI) fluorescence. Cells is the various phases of the division cycle are labelled.

Figure 4: This shows cell nuclei extracted from a paraffin block of a breast carcinoma, after fluorescence pulse-processing to eliminate cell clumps, as a histogram of PI fluorescence. Cycling diploid, as well as aneuploid tumour cells readily can be seen.

Back to DNA analysis.

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