Hi Brian, What stimulant are you using? Are you using a concentration that you know should give an appreciable change in fluorescence (not necessarily the same as a "physiologically stimulating" concentration)? If the answers to these are yes, it might be worth looking at your sheath buffer, Isoton II (and I think FACSflow) can suppress calcium responses to a range of agonists at quite low concentrations, e.g. if they get a chance to drip back into your sample while you're removing it from/replacing it on your machine. I found that putting the machine (Vantage or FACStar) in standby works fine, since it pinches the tube and stops drip-back, but using PBS as a sheath is easier and more reliable. Are you using a bench-top analyser or a big sorter? If the latter, it could be that the response is over by the time the cells reach the laser - try boosting after you replace the tube. Ray At 6:38 pm +0000 25/2/00, Newsom, Brian S. wrote: >We tried our first stab at calcium mobilation today and got dismal >results. >Following is the protocol we used: > >Preparation: >1. Make up fluo-3 and Fura Red at 10mg/ml in DMSO > >Procedure: >1. Wash cells in Ca++ Free media (HBSS)at RT. >2. Incubate cells in media containing 16uM fluo-3 + 40uM Fura Red for >45 minutes at 37C. >3. Wash cells once in Ca++ Free media (HBSS)at RT. >4. Resuspend in HBSS (Ca++ Free) at 2.0 X 10^6/mL >5. Run baseline analysis on Flow Cytometer collecting 30s-1min >of data. >Collect fluo-3 in FL1 and Fura Red in FL3. Collect both vs. Time. >6. Take sample off machine and add stimulus (leave machine in >run-continue collecting listmode). >7. Put sample back on and collect through sample peak (about 5 min). > >FL1 and FL3 were collected in linear. No flux was seen with either >dye. Any >suggestions on what we did wrong? Do we need HEPES in the buffer? Do >we need >different dye concentrations? Any suggestions welcomed and appreciated. > >Brian Newsom >Director, Flow Cytometry >Center for Cell and Gene Therapy >Baylor college of Medicine Ray Hicks ________________________________________________________________________ |University of Cambridge |Tel 01223 330149 | |Department of Medicine |Fax 01223 336846 | |Level 5, Addenbrookes Hospital |e-mail <rh208@cus.cam.ac.uk> | |Hills Road Cambridge |Web http://facsmac.med.cam.ac.uk | |CB2 |ftp server ftp://131.111.80.78 | |UK | | |_________________________________|_____________________________________|
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