Hi, I am a new user of the flow cytometry mailing list and I have enjoyed reading the postings over the last little while. I have found some of them very helpful! I guess now it is my time to try and seek some help! Our lab has recently begun investigating cyclin (specifically cyclins D1, D2 and B1) expression by flow cytometry. Our samples are MDA-MB-231 breast cancer cells and we simultaneously stain our cells with propidium iodide to look at DNA content. Our method and antibodies seem to be working but we have some questions about how to properly analyze our data. Since this is a new area of research for us I thought that some of you may be able to provide some insight into analysis for us. I have noticed in the literature (eg. Darzynkiewicz et al. Cytometry 25:1-13, 1996) that cyclin/DNA data is commonly analyzed by contour plots. I was just wondering if there is a reason for using contour plots as opposed to other methods such as dot plots or histograms (I realize histograms would be for cyclin expression alone, not with PI). In our lab we have done (up until now) mostly lymphocyte work and we are used to analyzing our immune cell data by dot plots when we do double and triple labelling. Can I analyze my cyclin/DNA data using dot plots? I have tried it using FL2-A (PI stain) versus FL1-H (FITC labelled cyclins). When I use dot plots I am having trouble knowing where to set the quadrant...S phase has to be either in the upper or lower quadrant and I don't know if this is correct. If anyone could offer any insight into proper analysis methods (we use Cell Quest) of cyclin/DNA data I would greatly appreciate it. Thank you in advance for your time, Lindsay Robinson Graduate Student Dept Agricultural, Food and Nutritional Science 4-10 Agriculture/Forestry Centre University of Alberta Edmonton, Alberta T6G 2P5 Phone: (780) 492-4240 Fax: (780) 492-9130
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