Hi Chris, What I usually do after the sorts is to take small amount of sorted cells (10-50µl, depending how generous researchers are), after 1 wash or at the same phase when they count and put them in a TruCount tube from BD. They have certain number (labeled on each pack, eg. 48500) of beads in lyophilized bead pellet. Making simple proportion you can, quite accurately calculate the number of your cells in the same volume as they are going to be proportionally distributed in your data file (mixing well is important). Don't forget to multiply the final number to get concentration per 1 ml of original tube (eg. 20µl x 50, 50µl x 20). In the same time you are checking the purities, after making exclusive gate on beads which are easily differentiated on fluorescence plots. If you can't get this tubes, you can make something similar by sorting 50000 of your alignment beads in an empty tube using COUNTER mode. The only thing with this method is that beads should be made fresh, requires more time, especially if you check recoveries on the same machine (I use FACScan for that) as you should rinse properly or you'll have >50000 of beads. It could also multiply possible error if your drops are not set correctly and you are not actually sorting 50000 beads. Hope this helps, Sasha. ---------------------- Dr Sasha Sreckovic Dept Path & Micro University of Bristol University Walk Bristol, BS8 1TD, UK Sasa.Sreckovic@bristol.ac.uk 0117-928-8606 On Thu, 2 Dec 1999 13:35:38 -0800 Chris lena <cali@liai.org> wrote: > > Hello gang, > My question deals with our FacStar. I am getting reports of counts that are > about 1/2 of the cell number the instrument says were recovered. I am > wondering if anyone has seen this or what to do to fix this problem. Cell > count is very important in many of our studies. > Thanks in advance and Happy Holidays, > > chris > > > > > > Chris Lena > Laboratory Technician > La Jolla Institute For Allergy and Immunology > phone:(858)678-4536 > fax:(858)678-4595 > cali@liai.org > http://www.liai.org > > _ _ > (_)-(_) > \"/ > =V= > > > >
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