At 18:03 12/04/99 MET, you wrote: > >Hello Flowers, >I would like to determine the oxidative burst of murine macrophages >and PMN by flow cytometry. In literature and the Orpegen test 123 >dihydrorhodamine is used, but there is no concentration mentioned. >Could anyone give me advice at which concentration to use it and how >to handle it ( I think it is extremely sensitive to oxidation-- do I >have to use inert gas?) >I would appreciate any comment on this subject. >Thanks, >Martin Lehner >University of Konstanz >Germany >lehner@tycho.gk-biochem.uni-konstanz.de > > Greetings Martin. I have used dihydrorhodamine 123 (Molecular probes cat. No. D-632) to monitor the oxidative burst in neutrophils and monocytes. i) Disolve 10mg of solid in 2.9ml DMSO and aliquote, store @-20'with desicant ii) Use at 10um with 1x10exp6 cells (you can also use it at this concentration in whole blood)* iii)Incubate at 37'C/15 minutes. iv) Tweak your cells with the agonist of choice eg fmlp etc iv) Run on flow *Add the stock solution to *warm* cell suspension -otherwise you may get the reagent precipitating out. I have used this technique for a couple of years and so far I have had no problems with the stored solution. You may find that in the whole blood system that the cells need to be 'primed' with something like GM-CSF before you see any response. Best wishes Arnold. _/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold pizzey Department of Haematology University College London 98 Chenies Mews London WC1E 6HX Tel: +44 171-209-6234 Fax: +44 171-209-6222 a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/ _/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold pizzey Department of Haematology University College London 98 Chenies Mews London WC1E 6HX Tel: +44 171-209-6234 Fax: +44 171-209-6222 a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/
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