>Dear All, > >I'm posting this for a colleague, so don't have all the details, but hope >that someone may have some suggestions. > >They are using "whole blood" bone marrow preparations in analysis of various >leukaemias/lymphomas, and consistently find a problem with autofluorescent >cells that give signals in both the FL1 and FL2 channels (they have that >typical non-specific diagonal look on a dot-plot of FL1-FL2). The >non-specific signals appear even in the absence of any antibody, so this >seems to be a true autofluorescence rather than non-specific antibody >binding. > >They are reluctant to gate out on FSC/SSC after back-gating as they feel >that they risk losing important cells. Does anyone have a simple way of >overcoming this problem, or even know for certain what the cells are likely >to be. They are not seen after ficoll separation, and scatter suggests that >they are of myeloid origin > > >Andy >(andy@serotec.co.uk) Hi Andy, a lot of people have that problem. What we do is use the compensation to get rid of the excess fluorescence in FL1 and FL2 by compensating into the FL3 channel. It's important not to compensate the autofluorescence into a FL channel you need for your analysis. You'll have to play around a bit with your settings using your controls; and it's a good idea to compare uncompensated versus compensated samples. I'd say the cells are macrophages; also multicolor staining might help to pull these cells out of the way. Ann
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:54:02 EST