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Selective determination of the doxorubicin content of individual acidic organelles in impure subcellular fractions

机译:选择性测定不纯的亚细胞部分中单个酸性细胞器中阿霉素的含量

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Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell fines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V-1 s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V-1 s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.
机译:由于细胞器制剂通常包含一种以上的细胞器类型(例如,酸性细胞器和线粒体),因此非常需要一种测量给定细胞器类型的特性同时避免由辅助亚细胞区室引起的偏差的技术。我们在这里报告使用毛细管电泳(CE)与激光诱导的荧光(LIF)双通道检测,以鉴定来自CCRF-CEM和CEM / C2细胞细粉的粗亚细胞级分中含有阿霉素(DOX)的酸性细胞器。如通过共聚焦显微镜证实的,酸性细胞器通过其荧光标记的纳米球的积累来鉴定。使用CE-LIF分析,可将单独检测到的细胞器分为三类:仅包含纳米球的酸性细胞器,包含纳米球和DOX的酸性细胞器以及其他包含DOX(例如线粒体)且没有可检测的纳米球的细胞器。在相同的CE-LIF运行中确定单个酸性细胞器和其他含DOX的细胞器的电泳迁移率,DOX荧光强度和纳米球荧光强度分布。酸性细胞器迁移率范围为(-0.7至-2.0)x 10(-4)cm(2)V-1 s(-1),而其他细胞器的迁移率范围为(-0.6至-3.5)x 10(- 4)cm(2)V-1 s(-1)。此外,通过校准检测器的响应,可以估算单个酸性细胞器和其他细胞器中的DOX含量。 CEM / C2和CCRF-CEM电池中每个酸性细胞器DOX的平均量分别为11.1 +/- 0.5和10.6 +/- 0.4 zmol。这份关于单个酸性细胞器中DOX积累的分析的第一份报告提出了一种分析酸性细胞器中荧光化合物积累的方法,该方法可用于研究酸性细胞器的成熟以及这些细胞器在耐药性中的作用。

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