From: Bernadette Bellette (bellette@utas.edu.au)
Date: Tue Sep 09 2003 - 17:39:19 EST
Hello everyone, I was hoping that someone could provide some advice to an honours student working within my department. I have copied and pasted her outline plus the problems she is having and if anyone has any advice she (and I) would greatly appreciate it! Many thanks in advance Bernadette Bellette PhD Student Discipline of Pathology University of Tasmania Hobart Tasmania Australia > > I am having a problem reading APC and PerCP together when using a 4 > colour system for identifying DC subtypes in peripheral blood. > > My staining combination is > HLA-DR:PerCP > CD11c:APC > lineage1 cocktail:FITC > CD123:PE > > Although it is clear that I have stained successfully in both > HLA-DR:PerCP and CD11c:APC, I am having dificulty getting a filter > combination that will adequately read both APC and PerCP together. > > The filter combination is: > FITC 525 band pass > PE 575 band pass > APC 630 long pass > PerCP 675 long pass > > The cytometer is: Coulter epics elite > > CD11c:APC staining is alone is clear as a bell using 675 band pass but > with 30 long pass used in 4 colour staining the APC parameter reads > high and the apparent staining correlates with 90 degree scatter > (occurs in negative and isotype control as well test tubes). > The actual staining with APC occurs at or below the intensity of the > contaminating light. > > We have used another filter combination (which I can't quote) > but this leads to contaminating light in the HLA-DR:PerCP parameter. > > Any suggestions?? > >
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