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1) If you are trying to measure the change in the average population intensity, then using
the ratio of the medians is a valid way to go.
2) Logamps aren't perfect, but the modern ones aren't "that" bad. We have done extensive
measurements on our BD machines (Facscan, Facstar+, Vantage) and find that the error in
their logamps is no greater than +-5%, except right at the lower and upper edges (the same
is true for the MoFlo - we have not looked at a Coulter machine in years, but would be
surprised if they are much different). So in a ratio, the error shouldn't be greater than
+-10%.
I am confused about your relative fluoescence scale. A four decade log amp should go from
1-10K (or .1-1K, etc.), not 0-10K. This distortion of the low decade could influence the
results.
3) In order to compare measurements from day-to-day on the same machine, standardization is
important. We standardize by setting a "standard particle" (i.e. bead) to the same position
on each channel for every experiment. This requires slightly different pmt voltages. The
relative sensitivity = 1/gain, where gain = (V/Vo)**7.4, where Vo is the standard pmt
voltage and V is the voltage used. This is an accurate measure of how well the instrument
is aligned overall. A 20% difference can indicate setup problems.
4) It is important to know the inherent error in the biological system. Do several
preparations of the same sample measured at the same time give the same results?
-Marty Bigos
Stanford Shared FACS Facility
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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