Polymorphonuclear leukocyte isolation from venous blood of the dog

Year 2019, , 75 - 78, 31.12.2019

Güneş Karakurt Kader Yıldız

https://doi.org/10.30704/http-www-jivs-net.621263

Abstract

The current study aimed to isolate neutrophils from venous blood samples of healthy dogs. Venous blood samples were obtained from Venae cephalica of clinically healthy dogs (n:5) into heparinized tubes. The blood samples (2 mL) and Percoll dilutions (45%, 54%, 63%, and 72%) prepared with Hanks Balanced Salt Solution were layered into sterile tubes. After centrifuge, the polymorphonuclear leukocytes (PMN) were aspirated between 63% and 72% interfaces of the Percoll dilutions into tubes. The samples of PMN observed under a light microscope. Viability was detected microscopically after stained with trypan blue dye. Diff-Quick staining was used to detect neutrophil purity of the isolated PMN. In the present study, the neutrophils ratio was calculated as 92% of the isolated polymorphonuclear cells. The neutrophil viability was calculated as 98% of PMNs isolated from the venous blood samples of healthy dogs. In the present study, the Percoll gradient centrifugation (72%, 63%, 54%, and 45%) is a fast technic for isolation of the neutrophils from venous blood samples of dogs.

 

Keywords

dog; venous blood; polymorph nuclear leucocyte; neutrophil; isolation

Supporting Institution

Scientific Research Coordination Unit of Kırıkkale University

Project Number

Project code: 2017 / 080

Thanks

This study was financially supported by Scientific Research Coordination Unit of Kırıkkale University (Project code: 2017 / 080).

References

• Bosco, A. M., de Almeida, B. F. M., Pereira, P. P., Narciso, L. G., Lima, V. M. F. & Ciarlini, P.C. (2013). High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro. BMC Veterinary Research, 9, 24.   Cools-Lartigue, J., Spicer, J., McDonald, B., Gowing, S., Chow, S., Giannias, B., Bourdeau, F., Kubes, P. & Ferri, L. (2013). Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. Journal of Clinical Investigation, 123, 3446-3458.
• Gosset, K.A.,MacWilliams P.S., Enright F.M. & Cleghorn B. (1983). In vitro function of canine neutrophils during experimental inflammatory disease. Veterinary Immunology and Immunopathology, 5, 151-159.
• Kaplan, M.J , Radic, M. (2012). Neutrophil extracellular traps: double-edged swords of innate immunity. Journal of Immunology, 189, 2689-2695. Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M, Bernuth H, Benarafa C, Roos D, Skokowa J, Hartl D (2015). Neutrophils: between host defence, immune modulation, and tissue injury. PLoS Pathogens, 11, e1004651.
• Lawson, C., Smith, S.A., O'Brien, M.& McMichael, M. (2018). Neutrophil extracellular traps in plasma from dogs with immune-mediated haemolytic anemia. Journal of Veterinary Internal Medicine  32, 128-134.
• Jeffery U, Gray R. D., & LeVine D. N. (2016). A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release. Journal of Visualized Experiments, 21(117),1-4.
• Li, R. H. L. & Tablin, F. (2018). In vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy. Journal of Visualised Experiments, 138.
• Li, R. H. L., Johnson, L. R., Kohen, C. & Tablin, F. (2018). A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy. BMC Veterinary Research, 14, 210.
• Marchi, L. F., Sesti-Costa, R., Chedraoui-Silva, S.& Mantovani, B. (2013). Neutrophil isolation protocol. Comparative Clinical Pathology 23(5),1469-1476.
• Mayadas, T. N., Cullere,X. & Lowell, C. A. (2014). The multifaceted functions of neutrophils. Annual Review of Pathology, 9: 181-218.
• Mosca, T. & Forte W.C. (2016). Comparative efficiency and impact on the activity of blood neutrophils isolated by Percoll, Ficoll and spontaneous sedimentation methods. Immunological investigations, 45, 29–37.
• Oh, H., Siano, B.& Diamond, S. (2008). Neutrophil Isolation Protocol. Journal of Visualized Experiments, (17), e745.
• Patel, P. & Chatterjee, S. (2018). Innate and adaptive immunity: Barriers and receptor-based recognition, immunity and inflammation in health and disease emerging roles of nutraceuticals and functional Foods in Immune Support , 3-13.
• Rosales, C. (2018). Neutrophil: A cell with many roles in inflammation or several cell types? Frontiers Physiology, 9, 113.
• Sano J, Oguma K, Kano R, Tsumagari S, Hasegawa A.(2004), Decreased apoptotic polymorphonuclear leukocyte rate in dogs with pyometra., Journal of Veterinary Medical Science, 66(1):103-105.
• Selders, G. S., Fetz, A.E.,Radic, M.Z. & Bowlin, G.L. (2017).An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration. Regenerative Biomaterials, 4, 55–68.
• Sheats, M.K., Sung, E.J., Adler, K.B.& Jones, S.L. (2015). In vitro neutrophil migration requires protein kinase c-delta (δ-PKC) mediated MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) phosphorylation. Inflammation, 38, 1126–1141.   Sursal, N., Cakmak, A. & Yildiz, K. (2018). Neutrophil isolation from feline blood using discontinuous Percoll dilutions. Tierarztliche Praxis Ausgabe K: Kleintiere - Heimtiere, 46, 399-402.
• Swamydas, M., Luo, Y., Dorf,M.E. & Lionakis, M.S. (2015). Isolation of mouse neutrophils. Current Protocol of Immunology, 110, 3.20.1–3.20.15.
• Wei Z, Hermosilla C, Taubert A, He X, Wang X, Gong P, Li J, Yang Z, Zhang X (2004) Canine neutrophil extracellular traps release induced by the apicomplexan parasite neospora caninum in vitro, front. Immunol., 31 October 2016  https://doi.org/10.3389/ fimmu.2016.00436
• Yildiz, K., Gokpinar, S., Gazyagci, A. N., Babur, C., Sursal, N. & Azkur, A.K. (2017). Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle. Veterinary Immunology and Immunopathology, 189, 1-10.
• Yildiz, K., Gokpinar, S., Sursal, N., Babur, C., Ozen, D. & Azkur, A.K. (2019). Extracellular trap formation by donkey polymorphonuclear neutrophils against Toxoplasma gondii. Journal of Equine Veterinary Science, 73, 1-9.