Hi, When you do your intracellular NO measurements, do you first prime the cells with arginine? And have you ever used L-NAME as your iNOS inhibitor? Thanks, Rich Hastings -----Original Message----- From: szunino@biologie.uni-erlangen.de [mailto:szunino@biologie.uni-erlangen.de] Sent: Thursday, February 21, 2002 6:27 AM To: cyto-inbox Subject: Measuring iNOS activity by FACS For iNOS activity, we are using DAF-FM diacetate (Molecular Probes, also Calbiochem and Alexis) to measure increases in nitric oxide levels by FACS in conjunction with specific cell-permeable iNOS inhibitors (preferential for iNOS, i.e. mercaptoethylguanidine (MEG) and 1400W). The inhibitors are important because there has been some question in the literature about whether DAF can also bind calcium. In our hands, we can inhibit fluorescence of DAF using inhibitors of iNOS, so we are fairly confident that DAF is really binding NO and not calcium. DAF can also be used in fluorometric assays for quantification. Best regards. Simon Watson wrote: Dear Colleagues Does anyone have experience measuring iNOS activity in murine or human macrophages by flow cytometry? Secondly, are there any antibodies suitable for flow to detect the murine iNOS enzyme within macrophages? With many thanks and best wishes Simon Watson MRC Centre for Inflammation Research Medical School University of Edinburgh Edinburgh Scotland Dr. Susan J. Zunino Dept. of Genetics University of Erlangen-Nuremberg Staudtstr. 5 91058 Erlangen Germany Tel. 49 9131 852-8784 FAX 49 9131 852-8526
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