Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.
Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.
Lagaay
A M
AM
Section of Gerontology, University of Leiden, The Netherlands.
Bosman
C B
CB
Van der Keur
M
M
Ligthart
G J
GJ
Schuit
H R
HR
Tanke
H J
HJ
Hijmans
W
W
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000943:Antigens, Differentiation / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D007694:Killer Cells, Natural / Q000276:immunology; D007958:Leukocyte Count; D008027:Light; D008214:Lymphocytes / Q000276:immunology; D008856:Microscopy, Fluorescence; D011961:Receptors, Fc / Q000276:immunology; D017452:Receptors, IgG; D012542:Scattering, Radiation
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D011961:Receptors, Fc; D017452:Receptors, IgG