Analysis of single-cell cultures by immunoaffinity capillary electrophoresis with laser-induced fluorescence detection
Analysis of single-cell cultures by immunoaffinity capillary electrophoresis with laser-induced fluorescence detection
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DOI：
10.1002/bio.645
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发表时间：
2001-03-01
期刊：
影响因子：
2.9
通讯作者：
Phillips, TM
中科院分区：
文献类型：
--
作者：
Phillips, TM
神经肽调节免疫活性正成为基础和临床科学中的一个重要问题，需要在单细胞水平上进行分析。开发了一种微量取样程序，用于研究神经肽刺激淋巴细胞中生物学重要肽的分泌，该程序基于与免疫亲和毛细管电泳（ICE）结合的微透析取样，并使用光纤光谱仪进行激光诱导荧光（LIF）检测和二极管激光激发。该系统证明检测限在高阿托摩尔（10（-18）mol/L）范围内，纯标准品，并能够监测分泌从一个单一的细胞随着时间的推移。使用这个系统，它是可能的区分四种神经肽对调节细胞因子的T和B细胞释放的影响。CB 4（+）淋巴细胞在用P物质（SP）和降钙素基因相关肽（CGRP）刺激后，细胞因子分泌比基线增加7.5倍。B细胞对CGRP和血管活性肠肽（VIP）刺激（增加5.5倍）有反应，但对SP无反应。这些变化发生在刺激后12-20 h，并且一旦达到峰值分泌，在实验期间保持在该水平。版权所有（C）2001约翰威利父子有限公司
Neuropeptide regulation of immunological activity is becoming an important issue in both basic and clinical sciences, necessitating the need for analysis to be performed at the single-cell level. A microsampling procedure has been developed for studying secretion of biologically important peptides from neuropeptide-stimulated lymphocytes, based on microdialysis sampling coupled to immunoaffinity capillary electrophoresis (ICE), with laser-induced fluorescence (LIF) detection using a fibre-optic spectrometer and diode laser excitation. The system demonstrated a limit of detection in the high attomole (10(-18) mol/L) range with pure standards and was capable of monitoring secretion from a single cell over time. Using this system it was possible to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines. CB4(+) lymphocytes demonstrated a 7.5-fold increase in cytokine secretion over baseline following stimulation with substance P (SP) and calcitonin gene-related peptide (CGRP). B cells responded to CGRP and vasoactive intestinal peptide (VIP) stimulation (5.5-fold increase), but not to SP. These changes took place 12-20 h post-stimulation and, once the peak secretion had been reached, remained at that level for the duration of the experiment, This system demonstrates the ability to perform high sensitivity measurements on microsamples of biological fluids. Copyright (C) 2001 John Wiley & Sons, Ltd.