We recently concluded a 2 year long study where one of the parameters measured was the oxidative burst of neutrophils/monocytes, assayed by conversion of rhodamine to its fluorescent form. There were two problems - nucleated RBC's and cell death in activation with PMA. The problems were so bad, we couldn't set a gate on even the neutrophils, because they were "engulphed" in the "RBC scatter cloud ". The kit for oxidative burst detection includes PI and we added it every time. However in my ignorance of the instructions, I didn't realize that the histogram showing how to set the PI signal on FL3 came from a program which could set an acquisition gate based on a histogram. DOH! So I acquired it right, but stored it wrong - should have raised the threshold for FL3. When analysing in cellquest, I saw the bad purity and reported "specimen errors" for over half the samples in the study... But fortunately I cought on to my mistake a few weeks ago, put everything in FlowJo and re-analysed it based on the FL-3 bright PI+++ cells only (in this setup the cells of interest would be the bright ones). Turns out that all those "specimen error"'s are fine now. I guess the lesson here is "read the kit instructions _before_ the study begins, Maciej!" :o) ===== `---------------------------------------------` | Maciej S. Simm | 525 E 68th Street | | Research Technician | Room N-805 | | Cornell Medical Center | Tel. 212.746.3428 | `---------------------------------------------` |www.cd4cd8.com | 917-734-6280 | AIM - XsimmX | `---------------------------------------------` __________________________________________________ Do You Yahoo!? Get personalized email addresses from Yahoo! Mail - only $35 a year! http://personal.mail.yahoo.com/
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