some people are stimulating T2 cells with peptide for 18 hrs and some people use Beta 2-macroglobuline and some protocol says incubate peptide with T2 cells for 18 hrs and then incubated with Brifieldin A for another 3 hrs before proceeding for Flow cytometry.
Which protocol is correct one for peptide stabilization assay?
If I understand correctly, what you want to examine is the cell surface stability of the MHC class I molecules upon addition of peptide. If that is the case, you can do several experiments: first, see whether just by adding the peptide overnight (without FCS, beta 2m, or BFA), the signal of MHC class I increases respect to the cells where you didn't add peptide. That will tell that the peptide indeed binds to MHC class I; then, you can add a combination of peptide and beta 2m, and you will see almost for sure an increase in the signal, because the binding of peptide and b2m is cooperative (even b2m alone with no peptide will increase the signal); finally, the real experiment is to test the resistance to endocytosis of MHC class I molecules upon addition of your peptide: the better the peptide, the longer the time you will see MHC class I at the cell surface. In that case, you have to do a Brefeldin A decay assay. To do so,you can pre-incubate your cells with your peptide of choice for different time points in the presence of 5 ug/mL Brefeldin A, and then FACS. Your controls are cells without peptide, and cells with a peptide of an affinity to MHC class I that you know beforehand. If your peptide is indeed good, you will see that after about 4h, the signal of MHC class I at the cell surface remains almost identical as compared to the time point zero.
If you have doubts, check our paper on MHC class I endocytosis; where we describe the whole protocol and the mechanistic basis of the assay.
I hope this is helpful!
Hi, Sebastian. I want to check the Peptide stability in T2 cell line. Presently we are stimulating the T2 cell line with 20 ug/ml for 18 hrs in incomplete RPMI medium. We are getting a satisfactory result but there are many modification in this protocol. some use beta2-macroglobulin or some use Brifieldin A also. I am confused which protocol to follow.
If I understand correctly, what you want to examine is the cell surface stability of the MHC class I molecules upon addition of peptide. If that is the case, you can do several experiments: first, see whether just by adding the peptide overnight (without FCS, beta 2m, or BFA), the signal of MHC class I increases respect to the cells where you didn't add peptide. That will tell that the peptide indeed binds to MHC class I; then, you can add a combination of peptide and beta 2m, and you will see almost for sure an increase in the signal, because the binding of peptide and b2m is cooperative (even b2m alone with no peptide will increase the signal); finally, the real experiment is to test the resistance to endocytosis of MHC class I molecules upon addition of your peptide: the better the peptide, the longer the time you will see MHC class I at the cell surface. In that case, you have to do a Brefeldin A decay assay. To do so,you can pre-incubate your cells with your peptide of choice for different time points in the presence of 5 ug/mL Brefeldin A, and then FACS. Your controls are cells without peptide, and cells with a peptide of an affinity to MHC class I that you know beforehand. If your peptide is indeed good, you will see that after about 4h, the signal of MHC class I at the cell surface remains almost identical as compared to the time point zero.
If you have doubts, check our paper on MHC class I endocytosis; where we describe the whole protocol and the mechanistic basis of the assay.
I hope this is helpful!
Thanks a lots for your detailed information Mr Montealegre. I got a clear idea what to do and how to do.
Dear Sebastian,
It would be so grateful if you could give me the link of the paper that describe the protocol of Brefeldin A decay assay.
Best regards
Dear Rukaia,
here is the link.
Article Dissociation of 2-microglobulin determines the surface quali...
Good luck!
Sebastian
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