Dear colleagues One of my colleagues has asked me to do flow cytometric analysis on eosinophils for his study. He had stained eosinophils with an antibody linked to Alexa Fluor 488 (exitation: 488 nm, emission 518 nm). I had not used this Alexa Fluor before and when I acquired the cells I found a shift in the peak in the attached chart: histogram C. He did not know that he should use isotype control, so isotype control was not used in these analysis. I want to give him a report about his results and I ask you to please tell me what you think regarding the following: 1. First thing, I think the eosinophils are not pure by looking at the distribution of the cells at histogram A. Do you think the population which I have gated in histogram A contains most of the eosinophils needed for the analysis? 2. The shift in the peak at histogram C: I will speculate that this may be due to sloppy staining methodology – perhaps poorly done wash steps, resulting in non-specific fluorophor binding. Alternatively, PMT voltage might have been too high during acquisition. Of course due to lack of isotype control, this could not be corrected. 3. Would you please help me identifying a commercial source for isotypic controls for Alexa fluor-labeled antibodies. Thank you. Fadia F. Mahmoud, Ph.D. Department of Medical Laboratory Sciences Kuwait University Faculty of Health Sciences 31470 Suliebikhat, Code 90805 State of Kuwait TEL: 011 965 482-4769 TEL: 965-950-0443. FAX: 011 965 483-3631 E-mail: fadiaphd2004@yahoo.com --------------------------------- Do you Yahoo!? Yahoo! Mail is new and improved - Check it out! This attachment - 'Flow data.jpg' - 223.98 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/64c92a28d1fb082d4ad55589aa574ee61ac60bca.jpgReceived on Mon Jul 12 13:18:01 2004
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