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
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