title: Non-activated T and B lymphocytes become morphologically distinguishable after detergent treatment.
abstract: T and B lymphocytes are difficult to distinguish morphologically even with electron microscopy, and antibodies are generally used to make the distinction. A specific reagent, consisting of nonionic and cationic detergents, is used for leukocyte differentiation using the Sysmex automated blood analyzer. This reagent increases cell membrane porosity and enables the introduction of fluorescent dye into leukocytes. In this study, we investigated the effect of this specific detergent on the morphology of T and B lymphocytes. T and B lymphocytes were obtained by density gradient centrifugation and magnetic cell sorting, with a minimum of 90% isolation efficiency. T and B lymphocytes were then treated with the specific detergent and fluorescent dye, and their distribution was analyzed based on side scatter and fluorescence intensity using general-purpose flow cytometry (FCM). Fluorescent images were observed using a confocal laser scanning microscope (CLSM), cellular inner structures using a transmission electron microscopy (TEM), and cell surfaces using a scanning electron microscope (SEM). The ratio of cholesterol to total lipid in cell membranes of B and T lymphocytes was measured using a fluorescent assay kit. The distribution of fluorescence intensity was different between T and B lymphocyte clusters, according to the FCM analysis. CLSM observations revealed that the fluorescent dye mainly stained cytoplasmic organelles. FCM, TEM, and SEM observations revealed that B lymphocytes are more likely to lose surface antigens and intracellular organelles than T lymphocytes, which allows the visual distinction between T and B lymphocytes. The ratio of cholesterol to total lipid in T lymphocyte membranes had tendency higher than that in B lymphocyte membranes. In this study, we demonstrate that cells with differences in cell membrane cholesterol amounts, such as B and T lymphocytes could be identified using an inexpensive detergent, as an alternative to costly antibodies.
authors: Cell Analysis Center, Scientific Affairs, Sysmex Corporation, Japan. Kono.Mari@sysmex.co.jp
authors: Mari
authors: M
authors: Kono
authors: Yuri
authors: Y
authors: Takagi
authors: Sawako
authors: S
authors: Kawauchi
authors: Atsushi
authors: A
authors: Wada
authors: Takashi
authors: T
authors: Morikawa
authors: Kunihiro
authors: K
authors: Funakoshi
mesh_terms: D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002784:Cholesterol / Q000032:analysis; D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008563:Membrane Lipids / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D015388:Organelles / Q000737:chemistry; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D003902:Detergents; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D002784:Cholesterol