Tierarztl Prax Ausg K Kleintiere Heimtiere 2018; 46(06): 399-402
DOI: 10.1055/s-0038-1677404
Kurzbericht – Short Communication
Georg Thieme Verlag KG Stuttgart · New York

Neutrophil isolation from feline blood using discontinuous Percoll dilutions

Isolierung neutrophiler Granulozyten aus venösen Katzenblutproben mit diskon tinuierlichen Percoll-Gradienten
Neslihan Sursal
1   Department of Parasitology, Ankara University Graduate School of Health Science, Ankara, Turkey
,
Ayse Cakmak
2   Department of Parasitology, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey
,
Kader Yildiz
3   Department of Parasitology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey
› Author Affiliations
Further Information

Publication History

Received: 07 June 2018

Accepted after revision: 10 August 2018

Publication Date:
18 January 2019 (online)

Summary

Objective: Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. Material and methods: Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63 %, 54 % and 45 %). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72–63 % Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. Results: The mean PMN number was 22 x 105 per ml (minimum – maximum: 20–26 x 105/ml). Neutrophil homogeneity was > 95 % in the cell suspensions. The viability of isolated neutrophils was > 98 %. The technique did not result in neutrophil activation. Conclusion and clinical relevance: Discontinuous Percoll gradients (72 %, 63 %, 54 % and 45 %) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.

Zusammenfassung

Ziel: In einigen Studien wurden in vitro neutrophile Granulozyten aus Katzenblut isoliert. Der wichtigste limitierende Faktor für diese Studien ist das geringe Blutvolumen, das sich ohne Entwicklung potenziell lebensbedrohlicher Komplikationen gewinnen lässt. In dieser Studie versuchten wir, neutrophile Granulozyten aus venösen Katzenblutproben mit diskontinuierlichen Percoll-Gradienten zu isolieren. Material und Methoden: Venöses Blut wurde aus der Vena cephalica von erwachsenen klinisch gesunden Katzen entnommen. Die Blutproben wurden auf Percoll-Verdünnungen geschichtet (72 %, 63 %, 54 % und 45 %). Nach dem Zentrifugieren akkumulierten die polymorphkernigen Leukozyten (PMN) als Bande zwischen den Percoll-Verdünnungen 72 % und 63 %. Die Gesamtzellzahl wurde manuell unter Verwendung eines Lichtmikroskops ausgezählt. Der Prozentsatz der neutrophilen Granulozyten wurde mikroskopisch nach Diff-Quik-Färbung bestimmt und die Lebensfähigkeit der neutrophilen Granulozyten mit einer 0,01 %igen Trypanblau-Färbung bewertet. Grundlage für die Bestimmung der Aktivierung war eine intakte Zellmorphologie der isolierten neutrophilen Granulozyten. Ergebnisse: Die mittlere PMN-Zahl betrug 22 x 105 Zellen/ml (Minimum – Maximum: 20–26 x 105 Zellen/ml). Die Reinheit an neutrophilen Granulozyten betrug in den Zellsuspensionen > 95 %. Die Lebensfähigkeit der isolierten neutrophilen Granulozyten lag bei > 98 %. Die Technik führte nicht zu einer Aktivierung der Neutrophilen. Schlussfolgerung und klinische Relevanz: Der Einsatz der diskontinuierlichen Percoll-Gradienten (72 %, 63 %, 54 % und 45 %) ist geeignet, um neutrophile Granulozyten aus venösem Katzenblut zu isolieren. Die Technik ließ sich einfach durchführen und die Neutrophilenaktivierung war minimal.

 
  • References

  • 1 Amulic B, Cazalet C, Hayes GL, Metzler KD, Zychlinsky A. Neutrophil func tion: from mechanisms to disease. Annu Rev Immunol 2012; 30: 459-489.
  • 2 Comazzi S. Evaluation of neutrophil function. In: Schalm’s Veterinary Hematology. Weiss DJ, Wardrop KJ. eds. Ames, USA: Wiley-Blackwell; 2010: 1114.
  • 3 Kobayashi SD, DeLeo FR. Role of neutrophils in innate immunity: a systems biology-level approach. Wiley Interdiscip Rev Syst Biol Med 2009; 1: 309-333.
  • 4 Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 2013; 13: 159-175.
  • 5 Lafrado LJ, Olsen RG. Demonstration of depressed polymorphonuclear leukocyte function in nonviremic FeLV-infected cats. Cancer Invest 1986; 4: 297-300.
  • 6 Morton D. Removal of blood from laboratory mammals and birds. Lab Anim 1993; 27: 1-22.
  • 7 Nathan C. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 2006; 6: 173-182.
  • 8 Oh H, Siano B, Diamond S. Neutrophil isolation protocol. J Vis Exp 2008; (17). pii: 745. doi: 10.3791/745.
  • 9 Quinn MT, DeLeo FR, Bokoch GM. eds. Neutrophil Methods and Protocols. Totowa, NJ: Human Press; 2007
  • 10 Rizzi T, Clinkenbeard K, Meinkoth J. Normal hematology of the cat. In: Schalm’s Veterinary Hematology. Weiss DJ, Wardrop KJ. eds. Ames, USA: Wiley-Blackwell; 2010: 813.
  • 11 Siemsen DW, Schepetkin IA, Kirpotina LN, Lei B, Quinn MT. Neutrophil isolation from nonhuman species. In: Neutrophil Methods and Protocols. Quinn MT, DeLeo FR, Bokoch GM. eds. Totowa, NJ: Human Press; 2007: 21-34.
  • 12 Strasser A, Kalmar E, Niedermüller H. A simple method for the simultaneous separation of peripheral blood mononuclear and polymorphonu-clear cells in the dog. Vet Immunol Immunopathol 1998; 62: 29-35.
  • 13 Tomazella GG, da Silva I, Laure HJ, Rosa JC, Chammas R, Wiker HG, de Souza GA, Greene LJ. Proteomic analysis of total cellular proteins of human neutrophils. Proteome Sci 2009; 7: 32.
  • 14 Toth TE, Smith B, Pyle H. Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats. J Virol Methods 1992; 36: 185-195.
  • 15 Udby L, Borregaard N. Subcellular fractionation of human neutrophils and analysis of subcellular markers. In: Neutrophil Methods and Protocols. Quinn MT, DeLeo FR, Bokoch GM. eds. Totowa, NJ: Human Press; 2007: 35-56.
  • 16 Vorobjeva NV, Pinegin BV. Neutrophil extracellular traps: mechanisms of formation and role in health and disease. Biochemistry (Mosc) 2014; 79: 1286-1296.
  • 17 Wardini AB, Guimaraes-Costa AB, Nascimento MT, Nadaes NR, Danelli MG, Mazur C, Benjamim CF, Saraiva EM, Pinto-da-Silva LH. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 2010; 91: 259-264.
  • 18 Weiss DJ, Kraemer R, Schmit K. Isolation of granulocytes and mononuclear cells from the blood of dogs, cats, horses and cattle. Vet Clin Pathol 1989; 18: 33-36.
  • 19 Yildiz K, Gokpinar S, Gazyagci AN, Babur C, Sursal N, Azkur AK. Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle. Vet Immunol Immunopathol 2017; 189: 1-10.