From: Sammee VanArsdale (samv@pharmingen.com)
Date: Tue Apr 28 1998 - 13:05:48 EST
Dear Dr. Baran, The use of NOK-1 for detection of human FasL by flow cytometry was originally described by Kayagaki et. al. (J. Exp. Med. 1995, 182: 1777-1783). Since that time, several other groups have published with the clone for flow, including one which you did not mention, but which demonstrates specific NOK-1 staining on monocytes. Surface staining: N. Zavazava and M. Kronke. 1996. Nature Medicine, 2 (9):1005-1010 Intracellular staining: Villunger et. al. 1997. Blood. 90 (1): 12-20 On Monocytes: Oyaizu et. al. 1997. J. of Immunol. 158:2456-2463. Using NOK-1 in our own laboratories, we have obtained good results with a three step indirect method, incorporating the metalloproteinase inhibitor, KB8301, for optimal detection. For PBMC's, we stimulate with 2 ug/ml ionomycin for three hours in the presence of KB8301 (10 uM), then stain with NOK-1 (purified format) at 0.5 to 1 ug/million cells, followed by biotinylated goat anti-mouse Ig. We use normal mouse serum to block non-specific binding before adding SAv-PE and another mAb for double staining; CD16 is a useful indicator for us. This data is presented in our 1998 Research Products Catalog. We find that the percent positive is significantly higher with the inhibitor than without, an observation which was originally published by Kayagaki et. al. in the reference listed above. There are several reagents which are commercially available for detection of human FasL. A recent discussion/comparison appearing in Science and published in full at Science Online's internet site (www.sciencemag.org/cgi/content/full/279/5359/2015a) may be of interest to you. In it, the specificity of NOK-1, as well other PharMingen clones, NOK-2 and G247-4, is demonstrated by several methods including flow cytometry, western blot and immunohistochemistry. The clone mAb33 and the rabbit polyclonal antibody, C20, which were used by Kiener et al. in the study you mention (J.Immunol. 1997. 159:1594-1598), are compared to NOK-1 and G247-4 in this series of articles. Please feel free to contact PharMingen Europe (Tel.: 49-40-53-28-4480) for technical assistance regarding the use of our reagents for human or mouse FasL. You may also request a copy of the Science articles which are described above if you do not have access to the internet. We hope this will be of help to you. Good luck with your work. Sammee VanArsdale, Ph.D. Product Manager, Cell Biology PharMingen Date: 4/27/98 2:03 AM From: Jarek Baran Dear Flowers, I have been trying to measure the expression of Fas Ligand on the surface of human monocytes. Unfortunately, monoclonals which I use (Pharmingen, clone NOK-1, biotin-conjugated) did not stain monocytes, neither in the presence of metalloproteinase inhibitor (Kanebo KB8301) nor in the absence. Morever, I was not succesful using this antibody even for intracellular staining. In the only one paper dealing with the measure of Fas Ligand intracellularly in monocytes which I found (Kiener et al. J.Immunol., 1997, 159:1594-1598) the authors used different monoclonals: directed against carboxyl- and amino-terminal regions of FasL obtained from Santa Cruz Laboratories or anti-FasL mAb (clone 33) from Transduction Laboratories. Does it mean that monoclonals from Pharmingen do not work with monocytes? If so, let me know about anydistributors in Europe of working anti-FasL mAb. Or maybe is there a trick I am missing? Thanks for any advice. Jarek Baran Ph.D. Department of Clinical Immunology and Microbiology Polish-American Institute of Paediatrics, 30-663 Cracow, Poland
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