Hi Joan, Many years ago I recall doing some PI DNA staining of megakaryocytes. One of the peculiarities of these cells is that they re-duplicate their DNA so that as well as there being 2n and 4n populations there are cells with 8n, 16n, 32n, 64n and even greater amounts of DNA. In terms of running the samples on a standard cytometer this means that you will have to analyse the DNA signal using a log amplification in order to get all the different peaks on scale. This makes the traditional anaysis of cells in G1/S/G2 very difficult and I recall that we just attempted to assess the number of cells at each DNA level. I think that I just used a standard 70% ethanol fixation followed by washing, RNase treatment and 50ug/ml PI. Hope that helps! Derek On Thu, 14 Jun 2001, joan Kalnitsky wrote: > I have a client who is interested in looking at the poidy level of her > megacaryocyte cell line. The first time she came to the lab she brought > her samples and had used Vindelov's. Her histograms looked just like cell > cycle histograms. Not surprising as Vindelove's is used for cell cycle. > She admitted that when she had looked at ploidy levels years ago she did > not use Vindelov's, but did use a PI solution. > Can anyone offer what PI protocol she should be using for poidy levels, > and if I should be approaching the set up of the Flow Cytometer in a > different manner than cell cycle work. I have a client who looks at the > ploidy level of plants, but I know that they have to digest the plant > material first so I don't think their protocol would be appropriate for a > cell line. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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