P Ganey | PubMed Health

PubMed1991

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.

Flow Cytometry↗

PubMed1990

Unique role of oxygen in regulation of hepatic monooxygenation and glucuronidation.

The purpose of this study was to evaluate the hypothesis that NADPH supply in intact cells is regulated by oxygen tension. This was accomplished by studying monooxygenation in perfused livers from Ah locus-responsive C57BL/6J mice, where rates of monooxygenation are high. Elevation of flow rate decreases the hepatic O2 gradient and increases O2 delivery to the organ. Under these conditions, rates of p-nitroanisole O-demethylation were 2-3 times higher in perfused livers from fed or fasted mice at high (10 ml/min) compared with normal (5 ml/min) flow rates. Rates of monooxygenation were directly proportional to oxygen tension (half-maximal rates occurred with approximately 400 microM O2). On the other hand, rates were independent of oxygen concentration in isolated microsomes where NADPH was supplied in excess. The decrease in rate due to diminished O2 concentration in the intact organ could not be attributed to hypoxia, because O2 tension in the effluent perfusate exceeded 50 microM even when influent perfusate was saturated with 25% O2 and ATP/ADP ratios were in the normal range. Thus, monooxygenation of p-nitroanisole in perfused mouse liver is dependent on oxygen tension. Similarly, glucuronidation of p-nitrophenol was oxygen dependent in the intact organ but not in isolated microsomes supplemented with UDP-glucuronic acid. Taken together, these data support the hypothesis that, at high oxygen tensions (e.g., in periportal regions of the liver lobule), mitochondrial activity is increased, which in turn enhances NADPH and UDP-glucuronic acid turnover, leading to accelerated rates of monooxygenation and glucuronidation in intact cells. In support of this idea, NH4Cl, which utilizes NADPH for urea synthesis, inhibited monooxygenation in the perfused mouse liver at high but not low flow rates. Thus, important phase I and II detoxification reactions are regulated indirectly by the hepatic oxygen gradient, via mechanisms involving cofactor supply, when cytochrome P-450 is not limiting.

Animals↗