Whole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2',7' dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial lipopolysaccharide (LPS) on phagocytosis.Blood from healthy donors was preincubated with log doses of bacterial LPS B (0.1-1,000 ng/ml) or sterile pyrogen-free saline at 37°C from 0-120 minutes. LPS increased both P and OB in a dose-dependent manner (up to 62 and 121%, respectively) at all time points tested, and this effect on P and OB could be detected even with no preincubation. This LPSinduced phagocytic activity could be blocked by the addition of polymyxin B (10 pglml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA at doses > 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re-emphasize the necessity of using pyrogen-free reagents in any study of neutrophil function. 0 1992 Wiley-Liss, Inc.

This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

The role of cholecalciferol, 25(OH) D3, and 1,25(OH)2 D3, as modulators of melanocyte function and proliferation has been examined. Topical application of 100 micrograms cholecalciferol to the pinnal epidermis of DBA/2J mice for 5 or 10 days increased the number of L-dihydroxyphenylalanine-positive (DOPA-positive) melanocytes and had a synergistic effect with a low dose of ultraviolet B light (UVB). Application of 1 microgram 1,25(OH)2 D3 had a transient effect on epidermal melanocytes. Addition of cholecalciferol to pure cultures of human melanocytes did not alter their tyrosinase activity (therefore, melanin synthesis) or growth rate even after 72 hours of treatment. However, treatment of similar cultures with 1,25(OH)2 D3 at a concentration equal to or greater than 10(-8) M suppressed tyrosinase activity but did not affect proliferation. The effect of 25(OH) D3 was similar to, but lower in magnitude than, that of 1,25(OH)2 D3. We attempted to demonstrate the presence of specific receptors for 1,25(OH)2 D3 in normal human melanocytes using the monoclonal antibody (Mo Ab) 9A7 gamma raised against the receptor for 1,25(OH)2 D3. Melanocytes were exposed to 9A7 gamma and to a secondary biotinylated Ab and analyzed by the fluorescence activated cell sorter (FACS). An increase in the specific fluorescent signal was constantly observed. By using the immunoblotting technique, we observed a major immunoreactive species that migrated in the 53-kD region in normal melanocytes. The size of this major immunoreactive species was smaller in melanoma cells than in normal melanocytes. This correlates with the finding that the former cells were unresponsive to cholecalciferol, 25(OH) D3, or 1,25(OH)2 D3 treatment. These results predict a direct role for 1,25(OH)2 D3 as an effector of normal melanocyte function.

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non‐pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non‐pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non‐pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non‐pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide‐related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.

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