Boissy R E, Trinkle L S, Nordlund J J
Department of Dermatology, University of Cincinnati College of Medicine, Ohio 45267-0592.
Cytometry. 1989 Nov;10(6):779-87. doi: 10.1002/cyto.990100616.
Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.
流式细胞术（FCM）已被广泛用于分析细胞的各种生物学特性。在本报告中，我们描述了一种方法，通过该方法使用FCM来确定色素沉着和无色素黑素细胞混合群体的光散射图谱，以及随后用于两种细胞类型的分选和分离。此外，通过流式细胞术比较了色素沉着和无色素黑素细胞中的相对过氧化物含量。研究了来自色素沉着对照和三种遗传上不同的白化病来源的培养禽黑素细胞。对色素沉着和无黑素黑素细胞混合悬浮液中的前向与侧向光散射进行FCM分析，区分出两个重叠的细胞群体。对这两个群体的分选表明，表现出较多侧向和最小前向光散射的群体主要是色素沉着的黑素细胞，相反，表现出较少侧向和较多前向散射的群体主要是无色素细胞。这两种黑素细胞类型在内源性过氧化物水平上也表现出差异。利用非荧光化合物2'，7'-二氯荧光素二乙酸酯（DCFH-DA）测量细胞两个亚群中的过氧化物细胞内含量，该化合物在细胞内被细胞内过氧化物氧化为荧光二氯荧光素（DCF）。无色素的白化病黑素细胞内源性过氧化物含量最高，而色素沉着严重的细胞过氧化物相关荧光则少得多。通过增加测定中使用的DCF浓度，可以增强这种DCF荧光的量。这些流式细胞术方法可用于分离和培养表达不同色素水平的黑素细胞亚群，并研究黑素及其前体与黑素细胞中氢和脂质过氧化物之间的关系。
