>From: Becky Kelly <Eak@medicine.wisc.edu> >Organization: UW Dept of Medicine >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Date: Fri, 3 Mar 2000 12:54:18 CST >Subject: measuring degranulation by flow >Priority: normal >X-mailer: Pegasus Mail for Win32 (v2.54) >X-PMFLAGS: 34078848 0 1 29130.CNM > > >1) Does anyone have experience evaluating degranulation of >eosinophils (or other granulocytes) by flow cytometric analysis? >(Using either scatter or intracellular stain) > >2) I received a lot of queries regarding a previous question, but no >"answers". Does anyone have any ideas? > >" I am measuring cell surface receptors for cytokines. How do I >know if a decrease in fluorescence is due to modulation of receptor >or to masking of the receptor by cytokines that are acquired in vivo? >I have tried, unsuccessfully, to detect surface-bound cytokine before >and after incubating cells with cytokine. Is there a way to >dissociate ligand-receptor complexes without altering the receptor in >the process?" > >Thanks-Becky Greetings Becky, In answer to question 2; I have had some experience in quantifying the human G-CSF receptor in neutrophils, in particular, I have looked at the rate of receptor downregulation upon binding to its cognate ligand under certain conditions. One strategy which you might consider is to strip ligand from receptor using a low pH wash step; I have included the protocol below. Reagents i) Phosphate Buffered Saline (no Ca/Mg) pH 3.0@ 0'C (pH with HCL) (Acid PBS) ii) Phosphate Buffered Saline (no Ca/Mg) 2% fetal calf serum, 0.1% Sodium Azide (PFN) Method All manipulations to be carried out at 0'C (i.e. on ice) i) Pellet 2 exp5 cells at 0'C ii) Resuspend pellet in 1 ml Acid PBS iii) Incubate exactly 2mins on ice iv) Add 50 (fifty)ml PFN v) Centrifuge 0'C 400rcf /15mins vi) Resuspend pellet in PFN to desired concentration You may want to omit the azide from the PBS if you wish to do further functional studies Best regards Arnold. _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 0207-209-6234 Fax: +44 0207-209-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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