Percoll不连续密度梯度离心法分离红螯光壳螯虾血细胞

建立了利用Percoll不连续密度梯度离心分离红螯光壳螯虾血细胞的方法,并对配制分离体系的缓冲液以及分离体系的密度组成进行了优化。结果显示,由20%、65%和100%的Percoll组成的分离体系分离效果最优。在转速为1 810 r/min的条件下离心20 min之后,可将红螯光壳螯虾血细胞分为SGC与GC 2个细胞层。经流式细胞术分析,发现细胞层纯度均在95%以上,细胞死亡比率低于1.5%,可用于后续的细胞功能分析。此方法简单有效,为后续研究螯虾的免疫防御机制及病害防治方法奠定了基础。

Separation of hemocytes of Cherax quadricarinatus by Percoll discontinuous density gradient centrifugation

we developed a method to separate the GC and SGC of Cherax quadricarinatus by using discontinuous Percoll density gradient centrifugation. The dilution buffer and the combination of density gradient were optimized. We found that the density gradient should be diluted in anti-clotting buffer to prevent hemocytes aggregation, and the discontinuous Percoll density gradient that consisted of 20%, 65% and 100% of Percoll provided the best result compared with others. After centrifugation at 1 810 r/min for 20 min, the hemocytes were separated into two layers, corresponding to SGC and GC, respectively. Flow cytometry analysis showed that the purities of both fractions were >95% and the cell mortalities were <1.5%. The isolated cells were in good status and could be readily used for further analysis. This simple and effective method will facilitate the study on the function of different hemocytes and thus provide information for disease control.