Repeatability of chemical cytometry: 2-DE analysis of single RAW 264.7 macrophage cells
Repeatability of chemical cytometry: 2-DE analysis of single RAW 264.7 macrophage cells
复制标题
DOI：
10.1002/elps.200700017
复制
发表时间：
2007-07-01
期刊：
影响因子：
2.9
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Sobhani, Kimia;Fink, Susan L.;Dovichi, Norman J.
本文用双向凝胶电泳法结合超灵敏的荧光检测技术，对RAW-264.7小鼠巨噬细胞系单个细胞的蛋白质和生物胺含量进行了研究。在进行2-DE分析之前，根据细胞周期对细胞进行分类。细胞周期处于G2/M期的细胞被吸入一维毛细血管并裂解。细胞内容物经荧光标记后，用毛细管筛分电泳法分离。380多个组分从一维毛细管转移到二维毛细管，在那里用ME KC进一步分离，并用激光诱导荧光检测。将四种电泳图共有的25个斑点与二维高斯面进行拟合，以确定斑点的位置、宽度和幅度。CSE活动度的RSD为1.0+/-0.6%。斑点位置的平均不确定度是CSE维度中平均斑点宽度的1.3倍。平均标准偏差为0.37+/-0.13 S，小于该维度上的平均斑点尺寸。现货产能为200。斑点幅度的RSD为50%，反映了成分表达在细胞间的巨大差异。
This report presents the use of 2-DE with ultrasensitive fluorescence detection as a chemical cytometry tool to characterize the protein and biogenic amine content of single cells from the RAW 264.7 murine macrophage cell line. Cells were sorted by cell cycle prior to 2-DE analysis. Cells in the G2/M phase of the cell cycle were aspirated into the first-dimensional capillary and lysed. The cellular contents were fluorescently labeled and first separated by capillary sieving electrophoresis (CSE). Over 380 fractions were transferred from the first-dimensional capillary to the second-dimensional capillary, where components were further separated by ME KC and detected by laser-induced fluorescence. Twenty-five spots common to the four electropherograms were fit with a 2-D Gaussian surface to determine spot position, width, and amplitude. The RSD in CSE mobility was 1.0 +/- 0.6%. The mean uncertainty in spot position was 1.3 times larger than the mean spot width in the CSE dimension. The average SD in MEKC migration time was 0.37 +/- 0.13 s, which is smaller than the average spot size in this dimension. Spot capacity was 200. The RSD in spot amplitude was 50%, reflecting a large cell-to-cell variation in component expression.