Glutamate aqueous solutions are notoriously unstable, particularly when glutamate is at concentrations below 1 mM because glutamate forms pyrrolidonecarboxylic acid. In the experiments described in our paper (1), we routinely used an adhesion buffer (i.e., RPMI 1640 supplemented with 0.1% BSA) that was filtered and stored for prolonged periods at 4°C. Such storage resulted in slight pH changes, as deduced by the more pink color of the adhesion buffer. Nevertheless, we used it as such because it never affected the viability of the cells, as was evaluated routinely by trypan blue exclusion or by the experiments themselves.
Thus, we assume that our ability to reliably and repetitively measure the effects of 10 nM glutamate described in our recent paper (1), as well as test a much wider range of 10−14–10−4 M glutamate and obtain a consistent dose-response curve for glutamate-induced T cell adhesion, as described in an earlier paper (2), is linked to the aging of the RPMI 1640 and to the disappearance of glutamate that originally was present at the unstable concentration of 136 μM (i.e., 136 micromolar).
The judicious comment of Dr. Poulopoulou (3) now warrants the obvious need to actually measure the glutamate levels in aged RPMI 1640 solutions or try to resuspend the T cells in a glutamate-free medium.