From: Joost Schuitemaker (j.h.schuitemaker@AMC.UVA.NL)
Date: Tue Nov 11 2003 - 07:19:31 EST
Hi, dendritic cells and intracellular staining of cytokines is our area of interest for years already. And one of the targets is IL-10. We put a lot of effort in this and until today we didn't better results than you show us now. IL-10 seems to be a cytokine that is very difficult to stain intracellularly. When you look at literature you will find the hybridoma you suggest (JES3-9D7) as one of the best choices. At this moment we use an other system to have a read out for IL-10 production at a single cell level. But we use this for T cells and monocytes! We never tested this for dendritic cells, but I don't see a problem to use this method. It is called the cytokine secretion assay (CSA) and it is available from Miltenyi Biotec. It can be used for all CD45+ cells. And it is available for mouse and human systems. You can find some of our results at my website: http://home.wanadoo.nl/flowcytometry/ Results in relation to dendritic cells you can find at http://home.wanadoo.nl/flowcytometry/dendritic_cells.htm and results regarding the CSA you can find at http://home.wanadoo.nl/flowcytometry/secretion_assay1.htm . Should you have any questions don't hesitate to contact me. Kind regards, Joost ____________________________________________________ J.H.N.Schuitemaker Laboratory Manager / Senior Research Technician Cellular Immunology Group Cell Biology & Histology Academic Medical Center P.O.box 22700 1100 DE Amsterdam The Netherlands Telephone +31 (0)20 5665068 FAX +31 (0)20 6974156 Internet: http://www.amc.nl http://home.wanadoo.nl/flowcytometry -----Original Message----- From: Ibtissam Abdul-Jabbar [mailto:iajabbar@cicr.uq.edu.au] Sent: maandag 10 november 2003 2:02 To: Cytometry Mailing List Subject: IL-10 in flow cytometry Dear All, Judy is a PhD student in our center, she is measuring number of intracellular cytokines in human DC, using BD cytofix/cytooerm kit, IL-10 is one of them. The clone used was JES3-9D7 (anti-human IL-10, PE). 7 days old human DC were purified and stimulated overnight (15hrs) with LPS + IFN-gamma, then treated with Brefaldine for 6hr and staining after that, we are using FacsCalibur for detection. Results showed only very small population is positive, we think that is not realistic and we should get better shift than this If anyone doing IL-10 using this method would you please confirm or advice Many thanks iaj Results is as follows
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