I am looking for a protein synthesis inhibitor for lymphocytes. I wish to stain T cells expressing TCR inducible protein with tetramers for detection of antigen specific cells. The tetramer is low affinity and therefore I have to stain at 37C. This might affect the expression level of the protein I analyze. I add sodium acid to slow down cell metabolism and I would like to include protein synthesis inhibitor. Can anyone recommend one?
Cycloheximide is generally used as an inhibitor of protein translation.
I think, your major problem is fast internalization of MHC-tetramer/TCR complex which takes place at 37C. To prevent it, try to use proteinkinase inhibitors, such as dasatinib. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052435/
Thanks! Actually I did mean to block protein synthesis, but it looks like it is worth testing dasatinib too. Isn't sodium azid good enough for blocking internalization?
Dear Ziv, sodium azide acts on live cells similarly to carbon monooxide or cyanides, blocking heme group in enzymes. At 37C this results to fast cell death, that may be critical in course of detection of T cells capable to MHC-tetramer binding. Staining with MHC-tetramers (especially with low affinity TCRs) requires sifficiently prolonged incubation time and elevated temperature, that is hardly compatible with the presence of sodium azide in the medium.
Ok, thanks! I will test the Dasatinib effect on MHC staining in my own hands. In the paper they recommend incubation for 20 min treatment with Dasatinib and 20-60 min with the tetramer both in 37C. Did this work for you?
Dear Ziv, I did not use MHC-tetramer staining before. In the part dealing with tetramer staining, I heared this from others. In my history, I was very interested in interactions of TCR with different ligands, natural and artificial. Especially, I was intersted in interactions of multiplet tetrameric peptides (fragments of MHC molecules) with TCR, but I worked in another experimental system (FTOC). I could see biological effects of this interaction, but my trials to detect this by FACS were not successful (may be, due to fast internalization). So, I watched MHC-tetramer history closely. In any case, I would strongly recommend not to use sodium azide in cell culture in 37C. Otherwise, expected effect will be increase in background staining.
Alternatively, isolate these T cells by sorting and leave them in culture for some time before doing your analysis. If your tetramer does not induce significant activation or if such activation does not affect your down-stream analysis, this may allow you to get away in relatively short break. If it is due to general protein degradation, then you should try proteasome inhibitors. I do not see why protein synthesis inhibitor should help
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