Ca2+Dear Hege,
The quantum yields of fluo-3 and Fura Red are vastly different - fluo-3 is brightest at saturating Ca++ (>1 uM, QY about 0.1) and Fura Red is brightest when Ca++ free (e.g. 10 mM EGTA, QY >0.05). I have seen some good data loading Fura Red AM at 5 to 20x the concentration of fluo-3 AM, but the dye loading ratio depends on cell type.
One suggestion is to try each dye separately to see if you get any signal change before trying the combination. This way you can independently adjust loading concentrations to get a meaningful ratio. Fluo-3 at 1 to 10 uM seems to give consistently good results in most types of cells, so it is likely that you are not getting enough Fura Red into the cytoplasm or it is compartmentalized over the long loading time and so is insensitive to cytoplasmic Ca++ flux. You might want to try loading for only 15 to 20 minutes and see if there is any improvement in the Fura Red signal.
If you optimize loading times/concentrations for Fura Red, the ratio method with fluo-3 will likely work. Microscopy is also a useful tool to see where the dyes are localized in the cell over time.
I hope this is of some assistance.
Best regards,
Michael Kuhn, President
Helix Research
Fluorescence Chemistry
mkuhn@helixresearch.com
www.helixresearch.com
----- Original Message -----
From: Fjerdingstad, Hege
To: Cytometry Mailing List
Sent: Monday, June 25, 2001 5:16 AM
Subject: Ca2+
Hi!!
I have some problem measuring Ca2+ with my Epics Elite. I load the cells for 45 min with Fura red and Fluo-3. After washing the cells I run them with an argon 488 nm laser. I try to stimulate both ECV-304 cells and MNC with PHA / FMLP or histamin without seeing anything. I'm looking for the ratio between Fura red and fluo-3 against time. Do anyone out ther have a procedure for staing and also for doing the calculation??
Regards
Hege Brincker Fjerdingstad
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