Dear Everyone, Three things:- 1) Magnetic beads and Flow Sorting: I have presumed that the beds (Miltenyi) would interfere with sorting by flow cytometry, even though they are absolutely titchy. Please say if you have sorted cells from a population containing magnetically-labelled cells, to good effect. 2) Magnetically labelled cells: When checking magnetically labelled and isolated cells for purity by flow cytometry, has anyone looked at loss of label? I was wondering whether or not there is a significant loss of label somewhere from the positive fraction in coming off the column or later. - I do everything at 4degrees and despite using the usual concentrations of conjugated second abs see a significant proportion of totally unlabelled cells in the fraction which was positively bound to the magnetic column. 3)Sorting time: Finally, When sorting a rare population from whole bone marrow, what kind of sort rate should be used? How stable could I expect "my" Vantage to be over the number of hours I'd need to use it? I am extremely impressed by the indication that other users seem to have a stable settup for many hours. I have never sorted for more than four hours. I'd need to run at least 100 million cells to get enough of my chosen population. Thank you for your attention. Carolyn Jefferiss Carolyn Jefferiss Ph. D. Pharmacy and Pharmacology University of Bath Claverton Down Bath BA2 7HY
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