Primary culture of L. vannamei hemocytes

Description

One difficulty when studying shrimp viruses is the lack of a stable host cell culture system. Sometimes insect cell culture systems can be used as alternative workarounds, but these systems cannot directly represent the actual situation in shrimp. In order to overcome this obstacle, we established a shrimp hemocyte primary culture platform. This platform allows researches to observe and manipulate the interactions between virus and host under conditions close to those that exist in a real world infection.

Brief protocol

Primary shrimp hemocytes cultures were prepared as described previously with some modifications. Hemolymph was collected from syringe that contained with cooled anticoagulant solution (2 % NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citratic acid, 10 mM EDTA). Seeding 65 µl hemolymph in 200 µl shrimp hemolymph cultured medium (2x Leibovitz’s 15 medium which contained 10% FBS, 1% glucose, 0.005% NaCl, 100IU/ml penicillin, 100IU/ml streptomycin and 1.25 µg/ml fungizone) and settled for 2 hrs onto a cover glass in each well of a 24-well plate, hemocyte monolayers were prepared.

References

1. Wang Z, Hu L, Yi G, Xu H, Qi Y, Yao L. (2004) ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene. Biochem Biophys Res Commun 325: 899-907.