Re: no subject (file transmission)
Gerald E. Marti (gemarti@helix.nih.gov)
Thu, 13 Apr 95 16:46:46 -0400
>Dear Tom: welcome to the world of Quantitative Flow Cytometry (QFCM). Bob
>Vogt et al recently published a chatper on this subject. To the best of my
>knowledge
short of radiolableing methods and single photon mesurements, there is no
published method for the independent determination of flurochrome per bead
if coupled to the surface. There is an excellent study from Japan by
Tadahiro Oonishi and N. Uyesaka (J Immuno Methods 84 (1985) 143-154; and J
Immuno Methods 115 (1988) 159-167. We had an opportunity to compare their
beads which can be dissolved in alkali overnight with quantitative recovry
of FITC to Abe Schwartz's beads. These experiments were a good lesson in
the concept of an effective F/P ratio. You don't get everything back that
you put in. Having used five generations of standards begining with cRBC
and fixed thymus nuclei and most recently QSC microbeads, I suggest that
you might want to figure out a way to work with FCM Standards, Inc perhaps
in the form of a CRADA. There are also the beads of Poncelet which I
understand are not available in the US. I am presently writing about QFCM
in B-CLL and would appreciate being kept abreast of your work. There are
at least 50 other references that Bob Vogt and I have collected and they
are in a gray bound folder somewhere. Will look for it. Also at the Fifth
Flow Cytometry Conf in Ottawa (oct '94), Frank Mandy had a session
(mini-symposium) on QFCM which I chaired and about a week ago a small group
met again in Ottawa under the leadership of Howard Shapiro and Frank Mandy
to discuss QFCM. I was unable to attend but I was informed of the
differences between antibody binding (AB) and antibody binding capacity
(ABC) and I don;t recall if "PMT shot noise" was discussed. Hopefully some
kind of report will come out of one or both of these meetings. Best of
luck in your studies.
I am coupling FITC, PE and PE-Cy5 to beads of various sizes. I would like to
>use these as standards of known number of fluorochromes per bead. I am
>looking for a method to approximate the number of fluorochromes per
>bead. Is it reasonable to run the soluble fluorochrome, trigger on
>fluorescence and plot the number of soluble fluorochromes versus the
>mean of the fluorescence histogram. And then use the mean fluorescence
>associated with a known number of soluble fluorochomes
>when I run the beads as a "mean equivalent soluble fluorochome" in order
>to assign an approximate # of fluorochromes to each bead?
>I have run the Flow Cytometry Standards beads along with mine but do not
>want to buy theirs for the number of studies that we would like to do
>and the range of fluorescence they offer is too narrow.
>(I do acknowledge the difficulty of determining antigen concentration
>per cell due to the antibody affinity, number of binding sites as well
>as the fluorochrome:protein ratio).
>
>Thanks,
>
>Tom McHugh
>Dept. Lab. Medicine
>Univ. Calif., San Francisco
>mchugh@labmed.ucsf.edu
Gerald E. Marti
Flow and Image Cytometry Section
Laboratory of Medical and Molecular Genetics
Division of Cell and Gene Therapies
CBER FDA NIH Bdg 29 Rm 502
8800 Raockville Pike
Bethesda,MD 20892
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
