Hi,
I usually go for PBS with anti-CD3 coating 24h prior to human CD4+ T-cell activation for further functionality test (proliferation, phenotyping, polarisation, etc.) That is what I saw other studies do.
Recently, my supervisor said that I should use 0.1 M Carbonate-Bicarbonate Buffer (pH 9.6) to replace the PBS for better binding. I thought this one was used for ELISA, not TC. I couldn't find other studies using the same method for TC work. So here I pose this question.
Does it make sense in the TC context? Thank you for your time.
Hello Zhihua Wu,
No, you shouldn't. Carbonate-Bicarbonate buffer (pH 9.6) can interfere with the binding of anti-CD3 antibodies to their target (CD3). The higher pH and ionic strength of bicarbonate buffer can alter the conformation of the antibody, potentially reducing its ability to bind effectively to the CD3 receptor.
For T cell activation, it becomes necessary that the antibody retains its full functionality. So, it becomes crucial to choose a buffer that doesn't negatively impact its binding affinity and stability.
While bicarbonate buffer is a standard coating buffer for ELISA plates due to its ability to promote protein adsorption, the requirements in this assay are different. Its potential to interfere with anti-CD3 antibody binding and stability makes it less desirable for coating applications involving anti-CD3 antibody. A gentler buffer like PBS is more appropriate as it is known to better preserve the antibody's functionality and stability for T- cell activation.
Best,
PBS is preferred for coating CD3, I and others have been doing it for long and never we did experience any difficulties in the cultures/assays. I agree with Malcolm Nobre about the points he mentioned.
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