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Fish Shellfish Immunol. 2019 Sep;92:871-880. doi: 10.1016/j.fsi.2019.07.018. Epub 2019 Jul 9.

Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood.

Author information

1
School of Animal and Veterinary Sciences, University of Adelaide, Australia.
2
Department of Immunopathology, SA Pathology at the Women's and Children's Hospital, North Adelaide, SA, Australia; School of Medicine, Robinson Research Institute and School of Biological Sciences, University of Adelaide, SA, Australia.
3
School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia; Centre for Cancer Biology, University of South Australia and SA Pathology, Frome Road, Adelaide, SA, 5000, Australia.
4
School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia.
5
Department of Immunopathology, SA Pathology at the Women's and Children's Hospital, North Adelaide, SA, Australia; School of Medicine, Robinson Research Institute and School of Biological Sciences, University of Adelaide, SA, Australia. Electronic address: antonio.ferrante@adelaide.edu.au.

Abstract

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.

KEYWORDS:

Asian seabass; Dihydrorhodamine (DHR)-123; Flow cytometry; Lipopolysaccharide (LPS); N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLF); Neutrophils; Phorbol myristate acetate (PMA); Tumor necrosis factor (TNF); Zymosan

PMID:
31299464
DOI:
10.1016/j.fsi.2019.07.018

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