Fluorescent measurement of nano scale particles.

Max Warncke max.warncke at uniklinik-freiburg.de
Mon Jun 11 06:05:21 EDT 2007


Dear all,

Can I use flow cytometry to analyze the coupling efficiency of a 
fluorescent dye to a 200nm dextran coated iron particle?

I coupled AlxFluor 647 hydrazide to a carboxyl containing bead by EDC 
(carbodiimide). After coupling I get a very strong signal in the APC 
channel, but I was not able to trigger the bead population. I tried use 
side scatter or fluorescence instead of forward scatter as trigger, but 
without success. Does anyone has a protocol for measuring fluorescent 
nano particles or a idea which method could be suitable for this?

I also coupled an antibody to the fluorescent beads, but then I had the 
same problems. If I incubate cells with the fluorescent bead I cannot 
completely separate the beads from the cells by washing. The beads which 
are still in the solution are producing a very strong background signal 
and I am not able to trigger only on the cells.

(I have attached an example: two cell populations. the B220 positive 
population has the specific receptor, the B220 negative does not. 
incubating only with a fluorescent coupled antibody doesn't produce a 
background signal).

I would be very happy about any suggestions.

Best,

Max



-- 
Max Warncke, Ph.D. (max.warncke at uniklinik-freiburg)
Department of Hematology & Oncology
University Freiburg Medical Center

fon: ++49.761.2707183
fax: ++49.761.2707177
-- 


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