A novel Eriocheir sinensis primary hemocyte culture technique and its immunoreactivity after pathogen stimulation

In order to advance immunological research, a modified primary cell culture method for hemocytes from Eriocheir sinensis (Chinese mitten crab) was established using L-15 medium lacking FBS and CO2 at 28 degrees C. Under these conditions, cultured cells were stably propagated for 30 days. To evaluate cellular activity, expression levels of certain critical immune-related genes, melanization of the hemocytes, and the phosphorylation status of key molecules in cell signaling pathways were analyzed. mRNA expression level of EsDWD1, EsLecG and EsToll2 was upregulated after LPS, PGN and GLU stimulation. Meanwhile, stimulated by Vibrio parahemolyticus led to the melanization of the hemocytes, and the melanization was more obviously with the increasing concentration of bacteria. Moreover, exposure to V. parahemolyticus significantly induced JNK, ERK and p38 phosphorylation, with the greatest extent of phosphorylation observed 30 min post-induction. Collectively, our results establish a technique for E. sinensis hemocyte primary culture and demonstrate appropriate immunological response, regulation and activity. Statement of relevance: We declare that we have no conflict of interest statement. (C) 2015 Elsevier B.V. All rights reserved.