Hello!
I have been working with Jurkat T Cells for flow cytometry.
I noticed that although Jurkat cells are T cells, their positive signals for CD3 antibodies were not so high. I've used 4-5 CD3 antibodies with different fluorochromes, but none of them were able to fully separate the negative (unstained) population from the positive (stained) population. When stained, the signal does shift to the right, but does not shift so dramatically that when I try to gate, some (30-40%) of the population still remain in the region where unstained cells sit.
Does this have to do with CD3 abundance in Jurkat cells?
I will ask the stupid question first:
Have you optimized your antibody concentrations?
Are you doing the FACS yourself? I am assuming whoever is doing the flow, they've set up controls for the fluorochromes the respective CD3 antibodies you have tested, if not, adjusting voltages on a clear CD3 negative/positive to see if you can tease that signal to be a bit stronger could be helpful. Sorry I can't be more helpful on the immunology aspect of your question.
Lewis Williams I initially began with the recommended amount normally 2-3 ul and have increased the concentration to 5 ul so far. Next time I try, maybe I should try to increase slightly more.
Vincent Cheng I've been adjusting the voltage using 50:50 FMO and Fully stained. But only slightly afraid of changing too much. Maybe I should go bolder as well.
I would dilute your antibodies as well. It is common that antibodies are too concentrated. This will lead to excessive and non specific binding. Which will increase the signal of your negative population. Visually this will merge your negative population, with the positive population.
For human cells I often dilute 1:10. Cells from mice often require even more dilute antibodies. It will also depend on your number of cells per sample.
All the best,
Seung Hyung Lee if you can spend some time, like 5-10 minutes playing with cells and stains, don't be afraid to crank the voltage up or down 100-200 and watch the populations move around the plot, a good FACS operator will always spend time doing this if possible but I understand if you're like the users who used my facility, you pay for the time but if the experiment/cells are super important, you want to know you're gating correctly. How many colours are you doing? Sometimes if the excitation of two or more fluorochromes are similar, there's bleedover and that could also be why you're not seeing a clean population.
Vincent Cheng
Hi Vincent!
I am using 6 Colors but added compensations to resolve the bleed-over.
Actually, when I used PBMC using the same panel, I had no issue, but with Jurkats, I am seeing some problem with CD3 antibody. I think I should try to increase the voltage to get better separation between the negative and positive populations.
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