I intend to block the MHC-II(I-A/I-E) on mouse antigen presenting cells in-vitro. Which technique will be the best?
That depends on which I-A/I-E you are using. We blocked I-Ab in vivo with Y3P antibody. We picked Y3P because it had been successfully used in vitro and in vivo before us. It is too long ago for me to recall the details. Of course, an antibody may work by depleting all the cells expressing its antigen in vivo while not blocking the actual interaction. If you are blocking other MHC class II molecule(s), I would start by identifying old TCR transgenics restricted by the class II molecule of your interest and then google scholar and pubmed searching for papers that use it in FTOC (fetal thymic organ culture. You should eventually find an appropriate antibody and concentration needed for blocking T cell recognition. You can test and confirm blocking by stimulating allogeneic CD8-depleted responders in a mixed lymphocyte reaction and seeing that proliferation at day 5 is blocked by the same antibody at the same concentration. I can help with more detail if that would be helpful.
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Hi Jeffrey,
Thanks for the vivid explanation and papers. However, blocking using an antibody is a good option. Does the Y3P antibody functions in preventing MHCII expression or it blocks the MHCII from presenting to TCR? Thanks
It will block the presentation. It may also result in killing of I-Ab cells in vivo, and may also result in internalisation of I-Ab, not quite sure. It is worthwhile to either locate papers that used it in vitro to block and/or check by blocking stimulation of an allogeneic (say, B10.D2) responders depleted of CD8+ T cells in an MLR. I hope that this is helpful.
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