No, we do not prime the cells. The leukemia lines I work with
appear to have a bit of NO without any encouragement from me (elevated
levels have also been observed in B-CLL). When we treat the cells with our
agents, there is an increase in NO and we can block this increase with MEG.
I am in the process of confirming this increase in nitric oxide using the
Griess colorimetric assay for total nitrate/nitrite to be sure it is real.
I have tried L-NAME, but it was causing some trouble (pH problems in the
medium, no difference compared to D-NAME, not so good). I'm not sure it
was even getting into our cells; they can be little pills sometimes. MEG
worked great however, and if you can believe the literature, it is
reasonably specific and a more potent inhibitor for iNOS than L-NAME and
L-NMMA (which also didn't work on our cells). I haven't used the 1400W
yet, but will soon. It should also be a potent inhibitor. Best regards.
>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: Cytometry Mailing List
>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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