Hi 1. The MACS beads are indeed tiny and do not interfere with sorting at all. 2. If you are checking the purity of your cells with an antibody recognizing the same epitope as the MACS beads, you will have problems as many or most of the epitopes will already be occupied with the antibodies attached to the beads. You need another antibody which does not compete with the first one. 3. My Vantage is stable over many hours with only minor adjustments of the amplitude knob to keep the phase and break-off in the same place. You may have to back-flush and perhaps do a nozzle flush every now and then to clear gunk from the nozzle depending on sample prep. If you are sorting rare cells one trick is to increase the sample rate up to about the same as the drop drive frequency, change the theshold to the fluorescent channel which you have the rare cell marker in, increase the theshold until all the negative cells disappear and change sort mode to enrich. If your original sample rate was 27K (with no turbo sort and 70u noz) and your population is 1% then after increasing the theshold the new sample rate will be about 270. With a three drop envelope your purity will be in the region of 33% if you sort all cells above theshold. You then spin then down and resort using normal theshold, rate and normal-R sort mode. When doing your enrichment sort you should make your sort windows generous, scatter will not be that tight. You should also make sure you have no lumps in your samples, hanging a filter off your sample probe is best. Good luck Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> Carolyn Jefferiss <prscmj@bath.ac.uk> - 8/8/2000 4:35 AM >>> 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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