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.
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* FACS Laboratory, FAX: (44) 0171 269 3100 *
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