Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines

Yehfang Hu, Kevin Maisey, Parasuraman Aiya Subramani, Fuguo Liu, Camila Flores-Kossack, Mónica Imarai, Christopher J. Secombes, Tiehui Wang

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)104-113
Number of pages10
JournalDevelopmental and Comparative Immunology
Volume88
Early online date18 Jul 2018
DOIs
Publication statusPublished - Nov 2018

Fingerprint

Oncorhynchus mykiss
Leukocytes
Erythrocytes
Cytokines
Fishes
Interleukin-1
Vaccines
Pathogen-Associated Molecular Pattern Molecules
Cytophagocytosis
Myeloid Cells
Allergy and Immunology
Mitogens
Research
Genes
Interleukin-6
Blood Cells
Neutrophils
B-Lymphocytes
Up-Regulation
Cell Count

Keywords

  • Rainbow trout Oncorhynchus mykiss
  • Peripheral blood leucocytes (PBL)
  • Hypotonic lysis of erythrocytes
  • Phagocytosis
  • Proliferation
  • immune response

Cite this

Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines. / Hu, Yehfang; Maisey, Kevin; Subramani, Parasuraman Aiya; Liu, Fuguo; Flores-Kossack, Camila; Imarai, Mónica; Secombes, Christopher J.; Wang, Tiehui.

In: Developmental and Comparative Immunology, Vol. 88, 11.2018, p. 104-113.

Research output: Contribution to journalArticle

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abstract = "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.",
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note = "This 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.",
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T1 - Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines

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AU - Maisey, Kevin

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AU - Liu, Fuguo

AU - Flores-Kossack, Camila

AU - Imarai, Mónica

AU - Secombes, Christopher J.

AU - Wang, Tiehui

N1 - This 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.

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N2 - 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.

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KW - Peripheral blood leucocytes (PBL)

KW - Hypotonic lysis of erythrocytes

KW - Phagocytosis

KW - Proliferation

KW - immune response

U2 - 10.1016/j.dci.2018.07.010

DO - 10.1016/j.dci.2018.07.010

M3 - Article

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JO - Developmental and Comparative Immunology

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SN - 0145-305X

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