That's a trick question. If you want to know the proportion of beads with any amount of SA attached, the answer is no -- a single SA conjugated to Ax568 can't be detected by standard FACS. If you are trying to distinguish beads that have a good number of SA attached (say, 100-300) from blank beads, the answer will be "maybe". That depends on whether you can reliably detect 200 nm particles by scatter -- the answer is most probably not, but you can try (use log-side scatter as a trigger). If the beads are sufficiently coated with SA, then you can trigger on the Ax568 fluorescence, and enumerate the positive beads. If all beads are intrinsically fluorescent or labeled (say with Ax488) then you could trigger on Ax488 and count the ones that also have Ax568 vs. those that have no Ax568 fluorescence. Years ago, we use to analyze subcellular organelles by FACS, using log- scatter as a threshold (you really need to use sheath fluid that has been ultracentrifuged), or triggered on nile-red fluorescence (NR goes into membranes) and quantify the other fluorescences... the organelles were clearly in the sub-micron range, but it's hard to know how small they got. Sorting is trivial if you can detect them. mr On Jun 26, 2008, at 10:17 AM, Timothy Overton wrote: > Dear All, > > I just has a request from someone working in my department which I > think is theoretically possible, but I wondered if anyone has tried > anything like it. They have nanoparticles coated in biotin, to which > are attached Streptavidin conjugated to Alexa 568. The size of the > particles is around 200 nm across. They asked whether flow cytometry > could be used to determine the proportion of particles with Strep- > Alexa attached, and then if they could be sorted. Has anyone done > anything like this? > > Thanks, > > Tim > > > ***************************************** > Dr Tim Overton > Lecturer in Biochemical Engineering > School of Chemical Engineering > The University of Birmingham > Birmingham B15 2TT > t: +44 (0) 121 414 5306 > e: t.w.overton@bham.ac.uk > >Received on Fri Jun 27 16:38:00 2008
This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 - 03:12:00 EST
