Are you sure you are compensated correctly? If you over compensating the PE out of the PE/Cy5 channel you would see what you describe, the brighter the PE the dimmer the PE/Cy5. Very bright stains can also be a bit problematic to compensate especially with log amps, you can make the CD38 stain dimmer by spiking it with unlabelled antibody. Try running samles uncompensated then using the automatic compensation in one of the analysis programs like FlowJo. Best Simon Monard FACS Lab Manager Trudeau Institute 154 Algonquin Avenue Saranac Lake NY 12983 ph 518 891 3080 Simon Monard FACS Lab Manager Trudeau Institute 154 Algonquin Avenue Saranac Lake NY 12983 ph 518 891 3080 >>> "Andrea Dewar" <andrea.dewar@imvs.sa.gov.au> - 3/10/04 3:20 AM >>> Dear flow group, Please see below a problem encountered by some of my colleagues. Please reply directly to Andrew Zannettino at andrew.zannettino@imvs.sa.gov.au. Many thanks, Andrea We recently performed the 3-colour flow cytometric analysis of bone marrow (BM)derived from a patient with the plasma cell disease, myeloma, using directly conjugated FITC and PE antibodies, and a variety of primary antibodies detected by sequential incubation with a biotinylated secondary and SAV-PE/CY5 (Beckman Coulter). The problem we have encountered centres around the fact that the level of PE/CY5 fluorescence seen when used in isolation is greater than when it is used in conjunction with the PE conjugated primary antibody. All our compensation controls appear to be well set and in the range for the conjugates used. An example of what we see is as follows: when using a mouse mAB to CXCR4 on BM (detected with PE/CY5), the mean fluorescence intensity (MFI) is approximately 28.5. However, when a PE or FITC-conjugated antibody to CD38 is used, the MFI of CXCR4 drops to 19.9. Is this a phenomenon related to CD38??? Or to our PE/CY5 conjugate as the level of PE/CY5 fluorescence also dropped when detecting other markers which we detected indirectly on these cells. Please note that CD38 expression is extremely high on myeloma plasma cells (4th decade of fluorescence). We look forward to hearing from anyone who can help. Regards, Andrew ZannettinoReceived on Thu Mar 11 16:18:00 2004
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