From: Marty Bigos (mbigos@gladstone.ucsf.edu)
Date: Wed Sep 17 2003 - 15:24:48 EST
At 7:15 PM -0700 09/16/03, Yoav Altman wrote: >Flowers, > >An investigator bringing me samples for calcium flux studies has >some questions about her data that I'm not able to answer. I'd >appreciate any help from the calcium wizards out there. > >We're running Jurkat cells loaded with Indo-1 on a FACSVantageSE >DiVa. I'm using 80mW of MLUV from a Krypton laser for excitation >and BD's 510SP to split the signal to my two UV detectors. UV1 and >UV2 have 405/20 and 424/44 filters respectively; nothing unusual, as >this all came from BD's calcium filter set. Voltages for the PMT's >are 580 (UV1) and 520 (UV2). > >When we stimulate the cells with OKT3 we see a nice increase in >violet (Calcium bound Indo-1) emission. The problem is that we >don't see a corresponding decrease in blue (unbound) fluorescence. >Do any of you have any ideas as to why we aren't seeing a decrease >in unbound Indo-1 fluorescence? > >Thanks, > >Yoav Your data looks fine (to me)!. The rotation of the signal is exactly what you should see with stimulation; this is a decrease in green and an increase in violet for the cells that rotated. It might be easier to visualize if you construct (in FlowJo) the ratio of Violet/Green. Marty > >Dot plots of cells before and after stimulation with OKT3. > > > >Time-course of the same data set. > > >-- >Yoav Altman >Manager, High Throughput Cell Analysis Core >The BURNHAM INSTITUTE >10901 North Torrey Pines Road >La Jolla, CA 92037 > >Voice: (858) 646-3100 xFLOW (x3569) >Fax: (858) 646-3167 >email: <yoav@burnham.org>
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