Protocol abstract
Nature Protocols 1, - 1522 - 1530 (2006)
Published online: 9 November 2006 | doi:10.1038/nprot.2006.250
Subject Category: Isolation, purification and separation
Quality assurance for polychromatic flow cytometry
Stephen P Perfetto1, David Ambrozak1, Richard Nguyen1, Pratip Chattopadhyay1 & Mario Roederer1
Abstract
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.
- 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.
Correspondence to: Stephen P Perfetto1 e-mail: sperfetto@mail.nih.gov
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