The bust test manual actually describes the general setup, therby mentioning the detection of the RH123 in the green fluorescence channel. Have you actually run the assay also staining with the CD4 only, w/o any of the other ingredients? Does it only happen when you stimulate with the bacteria or also in you use the fmlp? Regards Gerhard -----Original Message----- From: Maciej Simm [SMTP:simmmmer@yahoo.com] Sent: Monday, November 15, 1999 8:39 PM To: Cytometry Mailing List Subject: monos, monos, monos.... Hello everyone. First, a newbie dye-chemistry question: is the emission for Rhodamine 123 similar to PE or FITC or PerCP? Assuming the basic FACScalibur laser configuration ( I don't remember the wavelenghts). I tried to resolve monocytes using a perCP cd4 antibody. In "unstimulated" (as in background level of activation) PBMC's CD4 can resolve between lymphocytes and monocytes b/c of level of expression. I know there are more efficient ways of doing this 4ex. CD14 and CD33. But in this experiment (oxidative burst assay) I was not able to detect the FL3 signal at all! It was suggested thereafter that CD4 gets dropped during activation and that could be my problem. Has anyone tried adding a PerCP marker to the burst test to enhance gating? I would apprieciate any suggestions. Maciej ===== `---------------------------------------------` | Maciej S. Simm | 525 E 68th Street | | Research Technician | Room N-805 | | Cornell Medical Center | Tel. 212.746.3428 | `---------------------------------------------` __________________________________________________ Do You Yahoo!? Bid and sell for free at http://auctions.yahoo.com
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