 |
 |
 |
 |
 |
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
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web