Hello Brian, Too bad about your first run. A couple of thoughts: Why fluo and Fura?- I thought you had a UV laser (for Indo). If you want to use Indo, I can send you an applications note on the set up for the MoFlo. Oh- are you doing this on a scan? Did the cells load? Were you looking at only internal mobilization of calcium? The influx from external (media) to internal is a much larger flux, and easier to see. With calcium-free media, you're only able to see the internal flux- a lot smaller and shorter signal as I recall. You might want to retry the experiment using calcium containing media, and try to get a flux using an ionophore like a-23187 or ionomycin. That's the biggest signal you can get, and often helps with troubleshooting. Also, if fluo/Fura behaves anything like Indo, shorter loading periods sometimes work better than longer, since the dye starts to compartmentalize and then becomes less sensitive. To minimize this ,we load for 30 minutes at most, and then the cells go on ice to slow things down. When you're ready to run the cells, bring them back up to 37. You can look at the cells under a fluorescence scope to see loading- it should be diffuse, not punctate or granular. If you see structure in the staining pattern under the scope, it's probably too long of a loading. Regards, Peter Lopez > ---------- > From: Newsom, Brian S.[SMTP:BSNEWSOM@txccc.org] > Sent: Friday, February 25, 2000 11:38 AM > To: Cytometry Mailing List > Subject: Calcium Flux help > > 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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