Reproducible proliferative responses of salamander (Ambystoma mexicanum) lymphocytes cultured with mitogens in serum-free medium.

There are several reports that proliferative responses (tritiated thymidine incorporation (3HTdR)) of salamander splenocytes cultured with phytohemagglutinin-P (PHA) or concanavalin A (Con A) in 1% fetal bovine serum (FBS)-supplemented medium are either statistically insignificant or never approach the magnitude typically observed in similarly treated cultures of frog lymphocytes. The present study confirms these findings, but also reports highly significant and reproducible PHA-induced proliferation of axolotl splenocytes and thymocytes when the medium is supplemented with 0.25% bovine serum albumin (BSA) rather than 1% FBS. In one study, splenocytes from six of six axolotls cultured in BSA-supplemented medium displayed a dose-dependent response to PHA with stimulation indices (SLs) ranging from 4.2 to 14.1. In contrast, SLs of PHA-treated cells from the same animals, cultured in parallel in FBS-supplemented medium, ranged from 0.8 to 3.0. In a kinetic study (cells harvested from days 3-7), maximal proliferation in BSA-supplemented medium was noted after 5 days; cells cultured in parallel in FBS-containing medium were not responsive to the mitogen at any time point. Although axolotl splenocytes do not exhibit PHA-stimulated growth in FBS-supplemented medium, they are reproducibly stimulated in this serum-containing medium by phorbol 12-myristate, 13-acetate (PMA). This suggests that FBS may interfere with (or does not support) some early step(s) in lectin-induced signalling, rather than with proliferation itself.