I want to deplete CD3 T cell from PBMC, how can I do that without magnetic bead separation?
You could try FACS sorting your cells after labelling with an anti-CD3 antibody. You will affect cell viability though.
Hi Diana Baxter, thanks for your suggestion.
Diana Baxter While I want to deplete CD3 T cell by a simpler way, not by using antibody labling sorting. I mean that, can I stimulate T cells with excessive CD3 antibodies (>10μg/mL)to achieve the purpose of eliminating CD3 T cells？
Hello,
High levels of anti-CD3 will likely just tolerize the T cells, not deplete them.
I was going to suggest FACS cell sorting but an alternative you could use is the old standby; stain the cells with anti-CD3 antibody of an isotype that fixes complement then expose the cell population to reconstituted rabbit complement which will lyse the vast majority of the CD3 expressing T cells.
However, you should do FACS analysis at least once to see that both your antibody and source of complement are functional and that the CD3+ve cells are truly depleted.
I agree with Randall Scott Gieni's suggestion but remember an even more gentle technique. When you mix your PBMC suspension with washed sheep erythrocytes, rosettes around CD3 positive cells would be formed. By a simple low rpm differential centrifugation you could remove those rosettes and, of course the majority of T cells. The remaining cells in the supernatant layers can be collected and used for further experiments.
Building off of what Joseph said, you can use a microfluidic approach to separate T-cell rosettes. Here is a recent study that attempted something similar: https://pubs.rsc.org/en/content/articlelanding/2023/lc/d2lc00817c/unauth
To learn more about different types of microfluidic devices you can use for size-based separation, check out this article: Article Recent advances in microfluidic cell separation to enable ce...
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