Characterisation of Chinook salmon (Oncorhynchus tshawytscha) blood and validation of flow cytometry cell count and viability assay kit

Lulijwa, Ronald; Mérien, Fabrice; Burdass, Mark; Young, Tim; Meyer, Jill; Nguyen, Thao V.; Trembath, Caroline

doi:10.1016/j.fsi.2019.02.059

Cited by 21 publications

(16 citation statements)

References 90 publications

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“…The mixture was incubated for 5 min at room temperature (18°C). The viability profile of the isolated mononuclear cells was determined using the Muse Count & Viability Assay Kit on the Muse Cell Analyzer in duplicate readings, as previously described (Lulijwa et al ., ). For the positive control, dead cells were prepared via thermal treatment at 100°C for 5 min, and viability was determined using the Muse Count & Viability Assay protocols to set the gates for live and dead cells before sample analysis.…”

Section: Methodsmentioning

confidence: 97%

“…Fish PBMCs were isolated from 284 μl of blood, as previously described (Lulijwa et al ., ), using Histopaque sterile‐filtered, density gradient medium (1.077 g ml −1 ) (10771‐6X100ML, Sigma‐Aldrich, Auckland, New Zealand). The heparinized blood (284 μl) was diluted 1:1 with sterile‐filtered (40 μm) PBS (pH 7.4) in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Fish PBMCs were isolated from 284 μl of blood, as previously described (Lulijwa et al, 2019a) Cell Analyzer in duplicate readings, as previously described (Lulijwa et al, 2019a). For the positive control, dead cells were prepared via thermal treatment at 100 C for 5 min, and viability was determined using the Muse Count & Viability Assay protocols to set the gates for live and dead cells before sample analysis.…”

Section: Isolation Of Fish Pbmcsmentioning

confidence: 99%

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Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short‐term poly (I:C) challenge

Lulijwa

Mérien

Burdass

et al. 2020

Journal of Fish Biology

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Polyinosinic:polycytidylic acid [poly (I:C)] was administered in vivo to Chinook salmon (Oncorhynchus tshawytscha) post‐smolts to determine the immune responses on haematological and cellular functional parameters, including spleen (SP), head kidney (HK) and red blood cell (RBC) cytokine expression, as well as serum metabolomics. Poly (I:C) in vivo (24 h exposure) did not affect fish haematological parameters, leucocyte phagocytic activity and phagocytic index, reactive oxygen species and nitric oxide production. Gas chromatography–mass spectrometry–based metabolomics revealed that poly (I:C) significantly altered the serum biochemistry profile of 25 metabolites. Metabolites involved in the branched‐chain amino acid/glutathione and transsulphuration pathways and phospholipid metabolism accumulated in poly (I:C)–treated fish, whereas those involved in the glycolytic and energy metabolism pathways were downregulated. At cytokine transcript level, poly (I:C) induced a significant upregulation of antiviral ifnγ in HK and Mx1 protein in HK, SP and RBCs. This study provides evidence for poly (I:C)–induced, immune‐related biomarkers at metabolic and molecular levels in farmed O. tshawytscha in vivo. These findings provide insights into short‐term effects of poly (I:C) at haematological, innate and adaptive immunity and metabolic levels, setting the stage for future studies.

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Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Isolation Of Fish Pbmcsmentioning

confidence: 99%

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Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short‐term poly (I:C) challenge

Lulijwa

Mérien

Burdass

et al. 2020

Journal of Fish Biology

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“…Peripheral blood mononuclear cell (PBMC) isolation and purification was performed using our established protocol [ 67 ]. Briefly, 284 µL of whole blood (collected in lithium heparin BD-Microtainers ® ) was diluted 1:1 with sterile filtered (Whatman 40 µm (Cytivia; Vancouver, BC, Canada)) PBS (SF-PBS) (pH 7.4) and centrifuged (971 rcf; 20 min) over a layer of 682 µL Histopaque sterile filtered density gradient medium (10771-6 (Sigma-Aldrich; Auckland, New Zealand)) in 1.5 mL Eppendorf tubes.…”

Section: Methodsmentioning

confidence: 99%

“…PBMC viability assessments were performed via flow cytometry (Muse ® Flow Cell Analyzer (Merk; Darmstadt, Germany)) and a commercial assay kit (Muse ® Cell Count and Viability Kit (Merck)) using validated protocols for O. tshawytscha leukocytes [ 67 ]. To first establish correct Muse ® gating parameters, a PBMC sample (10 5 –10 6 cells mL − 1 ) from a single fish was used to prepare two matched suspensions of live versus dead cells.…”

Section: Methodsmentioning

confidence: 99%

Uncoupling Thermotolerance and Growth Performance in Chinook Salmon: Blood Biochemistry and Immune Capacity

et al. 2021

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Ocean warming and extreme sea surface temperature anomalies are threatening wild and domesticated fish stocks in various regions. Understanding mechanisms for thermotolerance and processes associated with divergent growth performance is key to the future success of aquaculture and fisheries management. Herein, we exposed Chinook salmon (Oncorhynchus tshawytscha) to environmentally relevant water temperatures (19–20 °C) approaching their upper physiological limit for three months and sought to identify blood biomarkers associated with thermal stress and resilience. In parallel, blood biochemical associations with growth performance were also investigated. Temperature stress-activated leukocyte apoptosis induced a minor immune response, and influenced blood ion profiles indicative of osmoregulatory perturbation, regardless of how well fish grew. Conversely, fish displaying poor growth performance irrespective of temperature exhibited numerous biomarker shifts including haematology indices, cellular-based enzyme activities, and blood clinical chemistries associated with malnutrition and disturbances in energy metabolism, endocrine functioning, immunocompetence, redox status, and osmoregulation. Findings provide insight into mechanisms of stress tolerance and compromised growth potential. Biochemical phenotypes associated with growth performance and health can potentially be used to improve selective breeding strategies.

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Polyinosinic:polycytidylic acid in vivo enhances Chinook salmon (Oncorhynchus tshawytscha) immunity and alters the fish metabolome

et al. 2020

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Characterisation of Chinook salmon (Oncorhynchus tshawytscha) blood and validation of flow cytometry cell count and viability assay kit

Cited by 21 publications

References 90 publications

Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short‐term poly (I:C) challenge

Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short‐term poly (I:C) challenge

Uncoupling Thermotolerance and Growth Performance in Chinook Salmon: Blood Biochemistry and Immune Capacity

Polyinosinic:polycytidylic acid in vivo enhances Chinook salmon (Oncorhynchus tshawytscha) immunity and alters the fish metabolome

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