Abstract
Natural killer (NK) cells mediate spontaneous cytotoxicity toward abnormal and tumor cells and rapidly secrete numerous cytokines and chemokine`s which promote subsequent adaptive immune responses. The presented study aimed to compare between different methods for NK evaluation and determination. Ten samples of healthy individuals were collected from Al-Zahraa University hospital and evaluated for phenotypic identification for NK cells. Isolation procedure of NK was performed in Clinical Pathology Department, Hematology Unit, Flow-cytometry lab, Al-Azhar University. Protocols started with isolation of peripheral blood mononuclear cells (PBMCs) from fresh human whole blood depending on Ficoll-Paque density gradient centrifugation. Other protocol depended on flow cytometry sorting technique. All experiments were carried out in accordance with the approved guidelines. Sorting procedure using FACS Calibur machine yielded 64.97 % purity before cells were cultured, while mixed Ficoll separation of whole blood showed purity 22.74%. Through our study, it is clear to us that the percentage of purity of NK cells using the flow-cytometry sorting technique is much higher than the percentage of purity of NK cells using the separation method dependent on Ficoll-Paque density technique, Therefore, through our study, we see that it is better to rely on the flow-cytometry sorting technique using the flow cytometry device and CD56 +CD16 and CD3 antibodies in order to obtain the best purity of the NK cells (CD56+ CD16 positive /CD3 negative population) for use in medical purposes and laboratory research.
Highlights
Natural killer (NK) cells, first identified in 1975, are innate effector lymphocytes that differ from T cells and B cells
They are commonly defined as a population of CD3-negative and CD56+positive cells (Spits et al, 2013; Walker, Barlow, & McKenzie, 2013).NK cells develop outside the thymus in various other tissues, and they do not express a rearranged T-cell receptor (TCR) (Freud et al.,2017)
Protocol started with isolation of peripheral blood mononuclear cells (PBMCs) from fresh human whole blood depending on Ficoll-Paque density gradient centrifugation
Summary
NK cells, first identified in 1975, are innate effector lymphocytes that differ from T cells and B cells. Human NK cells comprise 10–15% of circulating lymphocytes, and they are present in both lymphoid organs and nonlymphoid peripheral tissues (Artis & Spits, 2015; Cooper et al, 2001; Freud et al, 2017) They are commonly defined as a population of CD3-negative and CD56+positive cells (Spits et al, 2013; Walker, Barlow, & McKenzie, 2013).NK cells develop outside the thymus in various other tissues, and they do not express a rearranged T-cell receptor (TCR) (Freud et al.,2017). It is clear to us that the percentage of purity of NK cells using the flow-cytometry sorting technique is much higher than the percentage of purity of NK cells using the separation method dependent on Ficoll-Paque density technique, through our study, we see that it is better to rely on the flowcytometry sorting technique using the flow cytometry device and CD56 +CD16 and CD3 antibodies in order to obtain the best purity of the NK cells (CD56+ CD16 positive /CD3 negative population) for use in medical purposes and laboratory research
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