Perfetto Stephen P, Ambrozak David, Nguyen Richard, Chattopadhyay Pratip, Roederer Mario
Flow Cytometry Core Facility, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA.
Nat Protoc. 2006;1(3):1522-30. doi: 10.1038/nprot.2006.250.
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.
本方案概述了一个三部分的质量保证计划，用于优化、校准和监测用于测量标记有五种或更多荧光染料的细胞的流式细胞仪（这种做法称为多色流式细胞术）。该计划的初始步骤（系统优化）确保仪器的激光器、镜子和滤光片针对多种荧光信号的产生和传输进行了优化配置。为了确定每个荧光检测器的灵敏度和动态范围，然后通过在一系列光电倍增管（PMT）电压范围内测量荧光来校准系统，方法是确定PMT电压范围和线性度（步骤2-10）并验证PMT电压（步骤11-17）。最后，为确保性能一致，我们提供了随时间监测荧光测量的精密度、准确度和灵敏度的程序。本计划的所有三个方面都应在安装时或流式细胞仪光路发生任何变化时执行。然而，这些程序中只有少数需要定期进行。
