Rapid and high quality preparation of peripheral blood leucocytes (PBL) is important in fish immunology research and in particular for fish vaccine development, where multiple immune parameters can be monitored on the same fish over time. Fish PBL are currently prepared by density separation using Percoll or Hispaque-1.077, which is time consuming, costly and prone to erythrocyte contamination. We present here a modified PBL preparation method that includes a 20 seconds hypotonic lysis of erythrocytes and a subsequent separation of PBL from cell debris by a cell strainer. This method is simple, rapid and cost effective. The PBL obtained are similar in cellular composition to those prepared by density separation but have less erythrocyte contamination as demonstrated by FACS analysis and the expression of cell marker genes. Marker gene analysis also suggested that PBL prepared by hypotonic lysis are superior to those obtained by the gradient method in that some high-density cells (certain B cell types and neutrophils) might be lost using the latter. The PBL prepared in this way can proliferate in response to the T cell mitogen PHA, and both lymphoid and myeloid cells can phagocytose fluorescent beads and bacteria, with the latter enhanced by treatment with pro-inflammatory cytokines (IL-1β and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1β and IFNγ) in terms of upregulation of proinflammatory cytokine gene expression. Such data demonstrate the utility of this approach (hypotonic lysis of erythrocytes) for PBL isolation and will enable more studies of their role in disease protection in future immunological and vaccine development research in fish.
Bibliographical noteThis work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC, BB/N024052/1) under the Newton Fund RCUK-CONICYT Research Partnership. YH was supported by a PhD Studentship from the Ministry of Education, Republic of China (Taiwan). PAS was supported by the Newton-Bhabha PhD placement programme (268692473) and FL was supported by a Newton International Fellowship funded by the Academy of Medical Sciences (AMS, NIF004\1036). MI was funded by Fondecyt 1161015 and KM was funded by Fondecyt 11171057.
- Rainbow trout Oncorhynchus mykiss
- Peripheral blood leucocytes (PBL)
- Hypotonic lysis of erythrocytes
- immune response