Time interval gating for analysis of cell function using flow cytometry.
We propose a method which significantly shortens the time required for both the collection and analysis of data derived from multiple sample, flow cytometric kinetic assays. We have defined the term Time Interval Gating (TIG) to describe this method. TIG effectively allows one flow cytometer to concurrently monitor several samples over the course of a kinetic assay. Data for all samples are stored in a single FCS 2.0 compatible listmode data file which we refer to as the TIG data file. TIG is adaptable to most commerical flow cytometers. Standard listmode analysis software can be used to analyze the TIG data files and correlate any combination of tubes and/or time intervals from the assay. Results for the entire assay can be displayed on a single two parameter plot. This paper describes how TIG is applied to neutrophil oxidative burst measurement using a standard EPICS Elite flow cytometer. In this assay, 11 samples were each monitored for 30 min to identify the extent to which volatile organic chemicals (VOCs) inhibited the oxidation of DCFH in stimulated neutrophils. TIG makes the oxidative burst assay practical for high volume screening by reducing the overall flow cytometer and analysis time required by a factor of ten. In addition, TIG provides an organized approach to managing data acquisition on instruments equipped with automated sampling systems.
Durack
G
G
Purdue University Cytometry Laboratories, Purdue University, West Lafayette, IN 47907.
Lawler
G
G
Kelley
S
S
Ragheb
K
K
Roth
R A
RA
Ganey
P
P
Robinson
J P
JP
D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects; D009504:Neutrophils / Q000502:physiology*; D010084:Oxidation-Reduction; D016897:Respiratory Burst / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014050:Toluene / Q000494:pharmacology
D005452:Fluoresceins; C029569:diacetyldichlorofluorescein; D014050:Toluene; D006861:Hydrogen Peroxide; D013755:Tetradecanoylphorbol Acetate