Hi Brian, A few points based on what you wrote. 1. You are loading cells with the AM ester of the dyes, yes? Were the cells labeled at all? Do you have some protein (BSA, FBS...) or a dispersing agent (Pluronic F-127) in the labeling medium? 2. Your Flou 3 and Fura Red concentrations are very high and may be quenching your signal. Try lowering them to 10 uM (Fura Red) and 6 uM (Fluo 3) or even lower. 3. You probably want to have Ca++ in the medium, because the intracellular stores are not unlimited. I even label the cells in medium containing ca++. Good luck Lynn ************************************** Lynn Dustin, Ph.D. Assistant Professor Molecular Microbiology and Immunology St. Louis University School of Medicine 1402 S. Grand Blvd. St. Louis, MO 63104 Phone: 314-577-8415 Fax: 314-773-3403 Email: dustinlb@slu.edu "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 > > ------------------------------------------------------------------------ > > Part 1.2 Type: application/ms-tnef > Encoding: base64
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