4-anisyltetrazolium-blue and resazurin

Standard cell culturing on plastic plates (two dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. The three dimensional (3D) systems, composed of extracellular matrix and/or pure amino acids which form a scaffold for cells, are more accurate in this respect. 3D cultures were primarily developed for cancer cells but there is also a need for their application in studies on inflammatory leukocytes. Herein we describe our approach to study neutrophil-like cells in the 3D system. We describe measures taken to establish a neutrophil-like cell line (nHL-60) and selection of 3D scaffolds (PuraMatrix alone or enriched with collagen type I) for their culturing. We focus on challenges in measurement of neutrophil viability in 3D cultures and based on our data we suggest application of resazurin, rather than tetrazolium-based dyes or trypan blue exclusion, for evaluation of neutrophil viability.

Topics: Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Survival; HL-60 Cells; Humans; Neutrophils; Oxazines; Tetrazolium Salts; Tissue Scaffolds; Trypan Blue; Xanthenes

Here we show the results of comparing cell viability, cytotoxicity, and apoptosis assays for measuring the time- and dose-dependent toxic effects of tamoxifen on HepG2 cells. The quantitation of adenosine 5'-triphosphate (ATP), 5-(3-carboxymethoxyphenyl)-2-(4,5- dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) tetrazolium reduction, and resazurin reduction methods used to estimate the number of viable cells all showed a similar trend of decreased cell viability after longer periods of tamoxifen exposure to HepG2 cells. The release of lactate dehydrogenase (LDH) as a marker for cells with a compromised membrane and the increase in caspase-3/7 activity as a marker for apoptosis were both shown to increase using the same tamoxifen exposure conditions that caused a decrease in HepG2 cell viability. The longer the duration of exposure of tamoxifen, the lower the concentration required to kill or induce apoptosis in HepG2 cells. In contrast, there was no change in LDH release from HL-60 cells using conditions of vinblastine treatment that caused an increase in caspase activity and a decrease in ATP content, suggesting a difference in the mechanism of cell death between the two model systems. Both the density of parent stock cultures used as a source of cells to prepare assay plates and the density of cells per well in the assay plates were demonstrated to be factors than can influence the apparent potency of a toxin in viability, toxicity, and apoptosis assays. These results illustrate the importance of understanding the kinetics and mechanism of cell death of each in vitro model system as prerequisites for choosing the most appropriate assay method.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Caspases; Cell Count; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Endpoint Determination; Humans; Indicators and Reagents; L-Lactate Dehydrogenase; Luminescent Measurements; Oxazines; Oxidation-Reduction; Spectrometry, Fluorescence; Tetrazolium Salts; Xanthenes