From: Leary, James (jleary@utmb.edu)
Date: Tue Feb 10 1998 - 16:10:00 EST
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@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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