We have a lot of experience with pHi measurements using SNARF-1: 1) Use linear amplification 2) I strongly recommend thinking in terms of nm rather than PMT designation. i.e. we use 640nm/580nm for SNARF. Referring to "Fl2" etc is tacky in this context, at least to a flow purist. The emission spectra for SNARF are shown in the Molecular Probes catalogue. Check that you bandwidths are roughly centred on either side of the isobestic point. 3) Set up your high voltages using a dot display of 640nm emission versus 580nm. You want live cells on scale for both signals. If a cell is off scale the ratio calculation is apt to assign it a value of 1024 channel numbers, which will cause erroneous values for pHi, particularly if the other signal is on scale. Dead cells appear as a dim fluorescence population. 4) You should get CV's for the ratio peak of around 2-3% for unperturbed cells. David Hedley Ontario Cancer Institute/Princess Margaret Hospital Toronto
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