Separation of mononuclear leukocytes and polymorphonuclear leukocytes from equine blood.
Abstract: The present study describes a two step technique for the separation of mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from whole equine blood. First, the leukocyte rich plasma was obtained by sedimentation of erythrocytes in the undiluted blood. Subsequently, separation of the different populations of white blood cells was performed by centrifugation with different gradients overlaid with the leukocyte rich plasma. The optimal separation of the mononuclear cells was obtained by the centrifugation of the leukocyte rich plasma overlaying the gradient containing 24 parts of 9.5% ficoll and ten parts of 34% isopaque. The mononuclear leukocytes (95% lymphocytes and 5% monocytes) formed a monolayer band at the plasma-ficoll-isopaque interface and other blood cells migrated to the bottom of the tube. For the separation of mononuclear and granular leukocytes from the blood, the gradient containing 24 parts of 10% ficoll and ten parts of 34% isopaque was used. The separated monuclear leukocytes responded to stimulation with phytohemagglutin and viability of both mononuclear and polymorphonuclear leukocytes was not affected by ficoll-isopaque separation.
Publication Date: 1976-07-01 PubMed ID: 1087178PubMed Central: PMC1277767
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- Journal Article
Summary
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This study outlines a two-step method to separate two types of white blood cells—mononuclear leukocytes and polymorphonuclear leukocytes—from horse blood.
Research Methodology
- The researchers used a two-step technique for the separation of different types of white blood cells from equine or horse blood. These cells include mononuclear leukocytes (containing mostly lymphocytes and some monocytes) and polymorphonuclear leukocytes.
- The first step involved obtaining a plasma rich in leukocytes (white blood cells) by allowing the erythrocytes or red blood cells to settle down from undiluted blood. This process is known as sedimentation.
- The second step involved the separation of different white blood cell populations by utilizing centrifugation. In this phase, scientists overlayed different gradients on top of the leukocyte-rich plasma before performing centrifugation.
Finding the Optimal Separation
- The researchers found that the optimal method to separate the mononuclear cells involved centrifuging the leukocyte-rich plasma after overlaying it with a gradient that contained 24 parts of 9.5% ficoll and ten parts of 34% isopaque.
- Following this process, the mononuclear leukocytes formed a distinct layer at the boundary between the plasma, ficoll, and isopaque. Most of the other blood cells went to the bottom of the test tube.
Separation of Different Types of Leukocytes
- To separate the mononuclear leukocytes and the polymorphonuclear leukocytes from the blood, the scientists adjusted the gradient to contain 24 parts of 10% ficoll and ten parts of 34% isopaque.
- Triggers (in this case, phytohemagglutin) were used to stimulate the isolated mononuclear leukocytes, which responded well. This is important for physiological studies.
- The scientists also observed that the separation technique using ficoll-isopaque did not affect the viability of either the mononuclear leukocytes or the polymorphonuclear leukocytes.
Significance of the Study
- This research presents an effective method to separate specific types of white blood cells from equine blood. It could be immensely useful in veterinary research and clinical applications, especially those focusing on the immune response.
- Further, the retention of cell viability post-separation strengthens the reliability of this method in studies involving cell functionality.
Cite This Article
APA
Targowski SP.
(1976).
Separation of mononuclear leukocytes and polymorphonuclear leukocytes from equine blood.
Can J Comp Med, 40(3), 285-290.
Publication
Researcher Affiliations
MeSH Terms
- Animals
- B-Lymphocytes
- Centrifugation
- Female
- Horses / blood
- Leukocyte Count
- Leukocytes / cytology
- Male
- Monocytes
- T-Lymphocytes
References
This article includes 5 references
- Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.. Scand J Clin Lab Invest Suppl 1968;97:77-89.
- Carter EI, Valli VE, McSherry BJ. The separation of peripheral blood cells of the horse.. Can J Comp Med 1974 Jan;38(1):72-4.
- Targowski SP. Serum immunoglobulin, dermal response, and lymphocyte transformation studies in horses with chronic diarrhea.. Infect Immun 1975 Jul;12(1):48-54.
- McGuire TC, Banks KL, Poppie MJ. Combined immunodeficiency in horses: characterization of the lymphocyte defect.. Clin Immunol Immunopathol 1975 Mar;3(4):555-66.
- Carson CA, Sells DM, Ristic M. A method for separation of bovine blood leukocytes for in vitro studies.. Am J Vet Res 1975 Aug;36(08):1091-4.
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