Hi All,
I have been trying to optimize a panel for ovine lymphocytes CD3, CD4 and CD8 population using flow cytometry. In each experiment, large proportion of dead cells stains positive for CD3, CD4, and CD8 compared to the live cells population. I have repeated this experiment thrice, but the same result.
Please have anyone encountered similar challenges, and if there are recommended suggestion.
Kind regards
Henry
Hello Henry, How fresh are the cells you are using to optimise the staining panel? Have your samples been frozen?
How are you evaluating death in your samples? What level of viability is there in your samples before you start your staining procedure? Dead lymphocytes can still be labelled with antibody though the results will be unlikely to have the same resolution.
Try looking at some of the work done here:Article The association between female reproductive performance and ...
regards
The problem does not seem to come from your panel but with your cells dying. Make sure that your buffers are fresh and have the correct pH. Make sure that your preparation of the single cell suspension is not too harsh.
Thank you for your response Martin Waterfall and Louis-Charles Béland . My samples are fresh, not frozen. The viability of my sample prior to staining is about 90 %. I also did a whole blood stain as well to elimninate possibility of cell death due to RBC lysis before staining. I still have same result of large population of dead cells staining positive for lymphocytes markers, but this was not the same with myeloid lineage markers. I also used the flow cytometry staining buffer (i.e., PBS, FCS and EDTA) to resuspend cells before flow acquision. But during staining, I used PBS only, because of the staining dye (Fixable viability dye e780) used to stain cells for live/dead.
Please further suggestion will be very much appreciated
Hello Henry, As far as I can see you should have far higher viability in your stained sample. I have found that cells function can be significantly effected by EDTA, and avoid using or use at as low a level as possible(0.5-1mM). I would not expect that EDTA is causing your issue with viability but at the moment it would appear the one thing I do not include in handling ovine samples.
Hello again Henry, Just occurred to me - have you titrated the live/dead fixable stain or using it at the manufacturers recommended concentration? I have found you often(almost always!) find these concentrations will simply be so high that they appear to stain everything. If so try titrating 1:10, 1:100 and 1:1000 to get an idea of actual concentration required for optimal discrimination. These dyes are amide binding dyes not DNA dyes, designed to covalently bind to intracellular amines. However there are surface amines, too much dye availability may saturate everything or lead you to interpret live material as dead be reference to debris rather than live cells. Is your 10% 'live' cellular material suprisingly low FSC material? Can you upload a plot(s) of FSC versus ef780 staining? Sorry I missed this in the last message!
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