即将举行的网络研讨会:实现无需离心的流式细胞术样品制备工作流程的标准化
流式细胞术是一种利用单克隆抗体在细胞和分子水平对单个细胞或其他生物颗粒进行多参数、快速定量分析的方法。它能高速分析数万个细胞,并能同时测量一个细胞的多个参数,具有速度快、精度高、精度好等优点,是目前先进的细胞定量分析技术之一。流式细胞仪的主要组成部分是光源、液体流动路径、信号检测传输和数据分析系统。流式细胞术是临床检测外周血白细胞、骨髓细胞和肿瘤细胞的重要组成部分。
流式细胞术由三部分组成
1.液流系统,包括液流室和液流驱动系统;
2.光学系统,包括激发光源和光束收集系统;
3.电子系统,包括光电转换器和数据处理系统。
流式细胞仪的工作原理是使悬浮在液体中的弥散物通过荧光标记的细胞或颗粒通过样品池转移,同时通过荧光探测器捕获的荧光信号转换成代表前向散射角、侧向散射角和不同荧光强度的电脉冲信号,经计算机处理形成相应的点图,直方图和三维图像分析。流式细胞术所用的标本为单细胞悬液,可以是血液、悬浮细胞培养基、各种体液、新鲜实体肿瘤的单细胞悬液、石蜡包埋组织的单细胞悬液等。
流式细胞术的基本原理
1.使各种液体和悬浮细胞样品新鲜,尽快完成样品制备和检测;
2.对不同细胞样品进行适当的洗涤、酶消化或EDTA处理,去除杂质,使贴壁细胞相互分离,形成单细胞状态;
3.对于新鲜的实体肿瘤组织,可采用酶消化、机械分散、化学分散等方法获得足量的单细胞悬液;
4.包埋石蜡组织切成40-50um厚的几片蜡片,用二甲苯脱蜡入水,然后用上述方法制备单细胞悬液;
5.单个细胞悬浮液中的细胞数量不应少于10000个。
流式细胞术的应用:
1.该方法测定的细胞DNA变异系数小,一般小于2%;
2.DNA倍性分析;
3.荧光染料对细胞内蛋白质和核酸的定量研究
4.快速细胞分拣和收集;
5.医疗应用:免疫功能研究、各种干细胞检测、癌患者多药耐药、细胞功能和代谢动力学、血小板分析(心血管疾病)、流式细胞术和分子生物学;
6.应用于外周血内皮细胞测定、调节性T细胞等前沿领域。
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