We have a cell line derived from mouse muscle that we are trying to label with HO 33342 for cell cycle. It contains both diploid and tetraploid cells. We can see the 2 GO/G1 peaks on the HO histogram but the cells appear to be effluxing out the HO in a manner identical to other cell lines that we have which express p-glycoprotein. When we load p-gp positive cells with HO, we add verapamil at 50 uM to prevent the efflux and we get reasonable looking HO cell-cycle histograms. However, when we load these mouse muscle cell line cells with HO in the presence of verapamil, we end up with a histogram containing all the cells in one very wide peak that has a CV of 50% - we can no longer distinguish 2 DNA populations. The viability of the labelled cells remains the same as the unlabelled ones (85-90%). Does anyone have an idea what is causing this and how we can overcome it to sort viable diploid cells? Thanks -- Barbara J Taylor Facility Manager, FACS Core Lab Bldg 37 Room 6008 37 Convent Drive NCI, NIH Bethesda, MD 20892-4255 phone 301.594.6892 fax 301.496.8709 taylorba@pop.nci.nih.gov
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