Hi all!! I am having a problem getting a flux. Let me explain... We are using a Vantage for examining calcium flux in the mast cell line RBL2H3. The filter set up is as usual (405/20 in FL4, 530/30 in FL5, 505SP in between), I am getting a 10x rise in ratio using Jurkats with ionomycin. However, the mast cells give a rather weak flux, approximately 2x increase in ratio with ionomycin. The strange thing is that by confocal, there is a huge rise in intracellular Ca, and by fluorimetry using Fura the same thing happens. Has anyone had a look at these cells before? Anyone got any tips? We have thought that the cells may be temperature sensitive, but nothing happens at RT or 37C. Or does the detachment from the dish do something? (But the flux is fine in the fluorimeter)? We are loading with Indo for 10-60 minutes before harvesting the cells. If anyone has any suggestions, they would be gratefully recieved! TIA, Derek. **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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