Hi all,
Has anyone perform neutrophil depletion and adoptive transfer experiments?
To determine the function of one neutrophil receptor, I performed the following neutrophil adoptive transfer experiments. For the depletion of the neutrophils, CD45.1 mice were injected with 0.2 mg anti–Ly6G mAb (clone 1A8, BioXCell) by tail vein 24 hours before the adoptive transfer of neutrophils from CD45.2 mice. 24 h later, 1×10^7 bone-marrow derived neutrophils in PBS from CD45.2 mice were injected to CD45.1 mice via the tail vein. The proportion of peripheral CD45.2 neutrophils was measured by flow cytometry at the subsequent 24 h and 48 h, and surprisingly, almost no CD45.2 neutrophils were present.
I wondered whether it was because the residual antibodies were depleting CD45.2 neutrophils or because neutrophils had a short life span.
Thank you very much!
Yes. Both of those are likely. The life time of your average peripheral blood neutrophil is only about 12 hours. So if you measure only after 24 hours, you should have lost most of them. The life span of an antibody on the other hand could be much longer. So that could well still be around and knock out your new neutrophils as soon as you put them in. Measure all the things, and you will see.
Hi,
Maybe try to stain your BM-derived neutrophils with Zombie Dye Agua or PI to make sure whether they are alive when transferred to the recipient.
A second point is you could try parabiosis, instead of the adoptive transfer to achieve your goal. In that case, the recipient will receive donor-derived neutrophils all the time.
Regards!
Qian
Qian Wu
Hello, Dr. Qian
Thanks for your constructive suggestion. The parabiosis you mentined, do you mean that two mice are surgically linked together so that they share the same blood circulation? This experiment sounds difficult.
Best wishes!
Fang Yafei
John Schloendorn
Dear Dr. Schloendorn,
Thank you very much for your suggestions. I will continue to review the literature and improve the experimental procedures.
Best regards!
Fang Yafei
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