Quality assurance for polychromatic flow cytometry
Quality assurance for polychromatic flow cytometry
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DOI：
10.1038/nprot.2006.250
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Roederer, Mario
中科院分区：
文献类型：
--
作者：
Perfetto, Stephen P.;Ambrozak, David;Roederer, Mario
本方案概述了一个由三部分组成的质量保证计划，用于优化、校准和监测流式细胞仪，该流式细胞仪用于测量用五种或更多种荧光染料标记的细胞（称为多色流式细胞术的实践）。该计划的初始步骤（系统优化）确保仪器的激光器、反射镜和滤光片经过最佳配置，以生成和传输多个荧光信号。为了确定每个荧光检测器的灵敏度和动态范围，然后通过测定光电倍增管（PMT）电压范围和线性（步骤2-10）并验证PMT电压（步骤11-17），在一定范围的光电倍增管（PMT）电压上测量荧光来校准系统。最后，为了确保性能的一致性，我们提供了监控荧光测量随时间变化的精密度、准确度和灵敏度的程序。本程序的所有三个方面都应在安装时执行，或者在流式细胞仪光路发生沿着变化时执行。然而，这些程序中只有少数程序需要在常规基础上执行。
This protocol outlines a three-part quality assurance program to optimize, calibrate and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (a practice known as polychromatic flow cytometry). The initial steps of this program (system optimization) ensure that the instrument's lasers, mirrors and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of photomultiplier tube (PMT) voltages by determining the PMT voltage range and linearity (Steps 2-10) and validating the PMT voltage (Steps 11-17). Finally, to ensure consistent performance, we provide procedures to monitor the precision, accuracy and sensitivity of fluorescence measurements over time. All three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer's optical path. However, only a few of these procedures need to be carried out on a routine basis.