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Mark, your analysis of FACScalibur vs. XL is not completely accurate.
The XL can use Cy5PE together with PE and FITC. Cy5PE is actually
even more sensitive than PE for low density antigens. Thus, both
machines can do 2 colors of high sensitivity. The FACScalibur can't
do Cy5PE and APC together very well, because of the substantial
cross-excitation. (Yes, I know that applications can be designed
where this "feature" will not hinder the experiment).
By the way, it is not energy transfer which is relevant to the dyes
FITC, PE,
PerCP, and APC, but quantum efficiency. Energy transfer refers to
tandem dyes such as Cy5PE. Also, it is not appropriate to compare
emission intensity at
"equivalent excitation" intensity for APC and the other dyes, since
they are different lasers. The real comparison is how bright a
reagent is compared to autofluorescence--a ratio which varies with
excitation intensity but can be optimized for some of the dyes.
And while the FACScalibur can sort, this capability is designed
primarily for rare-cell applications and is not suitable for most
sorting applications; this advantage over the XL may not be
significant to may laboratories.
Please note that I am not addressing the relative merits of the two
instruments.
mr
MARIO,
1. Quantum efficiency is a measure of energy transfer.
2. The FACScalibur can run CY5 without using PE as a tandem.
3.PE/CY5 conjugates have two major problems, one, every conjugate
has to be individually compensated, unlike PerCP, and two, CY5 is
nonspecifically taken up by cells of myeloid origin. (APC requires
no compensation when used with a second laser)
4,.The sorting capability on the FACScalibur works extremely well for
molecular applications in our laboratory. We sort cells for PCR, and
cytospins for FISH. I would never use the FACScalibur for rare cell
applications. These are reserved for the FACStar Plus. The
FACScalibur works very well for sorting most populations > 10% of
total to 99% purity.
5. In my experience, nothing is more sensitive for low density
antigens than PE, as it requires minimal compensation when used with
FITC, PerCP and APC. Sensitivity is limited only when using PE with
PE tandem conjugates such as PE/Texas Red and PE/Cy5. This is the
very reason a second laser to excite APC is so desirable.
6. PE>APC>PerCP>FITC>Autofluorescence!
Please note that I am not addressing the relative merits of the two
instruments.
Heads up,
Mark
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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
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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