Labeling cells with beads

Leary, James jleary at utmb.edu
Tue Feb 10 16:10:00 EST 1998



Calman -- No, there aren't many articles on this subject. Noel Warner and   
I did a lot of work in this area at Los Alamos back in 1978, but for a   
variety of reasons unrelated to the science (he soon went to BD and I   
soon left for Rochester) it never got published. We were trying to do a   
light scatter assay for phagocytosis. The take-home message is that small   
beads were detectable by side scatter but only for reasonably   
homeogeneous cell populations. These were beads in the range of about 1   
micron diameter. Jim Cupp and I subsequently used fluorescent immunobeads   
for detecting fetal Rh+ cells in maternal blood, and it worked very well   
(Cupp et al., Cytometry 5: 138-144, 1984). Fluorescent immunobeads work   
great provided they are not phagocytosed, but you do get steric hindrance   
problems, so your staining may not be stoichiometric. Unfortunately, as   
you cut down on the cross-sectional area of the beads to solve the steric   
hindrance problem, the fluorescence decreases as the cube of the   
diameter. At some point it becomes a losing game below about 0.3 micron   
if you want to detect a single bead. When delayed fluorescence beads   
become commercially available someday this will problem not be a problem   
since we will be able to pack maybe a 1000 times as much dye per bead   
without self-quenching problems. Until then you will fight the cross   
sectional area vs. volume problem. On a slightly different but still   
related subject you can grow cells on microbeads (one cell per bead) and   
distinguish rather easily between cells, beads and cells bound to beads   
(Sterner and Leary, Cell Biophysics, 15: 159-171, 1989). This may be   
useful to people studying cell adhesion molecules and can't use proteases   
to get a single cell suspension. We were looking at fibronectin. Good   
luck!   --- Jim Leary
 -----Original Message-----
From: Calman Prussin [SMTP:CPRUSSIN at atlas.niaid.nih.gov]
Sent: Tuesday, February 10, 1998 2:09 PM
To: cyto-inbox
Subject: Labeling cells with beads


I am interested in the possibility of using antibody labeled beads to
identify and exclude a population of cells using scatter. In this way I
could accurately exclude the unwanted cells from my analysis , but not
use up a fluorescence channel in the process. Another possibility would
be to use some of those amazingly fluorescent beads that go into the 4th
decade, beyond what my cells are capable of. A Medline search on  "flow
cytometry, beads"  and either "scatter", "exclusion" or "gating or
gated" yielded no relevant papers.

Ideas? Experiences? References?

Thanks,

Calman Prussin



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