Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique.
We describe the liquid-overlay technique which provides an ultra-low attachment surface on which PRHs can be cultured as spheroids. © 2019 The Authors. Basic Protocol 1: Preparation of agarose-coated plates Basic Protocol 2: Primary rat hepatocyte isolation procedure Basic Protocol 3: Primary rat hepatocyte spheroid culture Basic Protocol 4: Immunofluorescent analysis of PRH spheroids.
PMID: 31529797 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research.
Authors: Taylor CA, Shawlot W, Ren JX, Mukhopadhyay S
Abstract
Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system. The first protocol describes how to use gene targeting in mouse embryonic stem cells to generate mice with conditional alleles. Subsequent protocols describe how to recover Cre transgenic mice from cryopreserved sperm using in vitro fertilizat...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Isolating Rat Intestinal Explants for In Vitro Cultures.
Authors: Kothari A, Rajagopalan P
Abstract
The small intestine is an important organ primarily involved in digestion of food and absorption of nutrients. In vitro intestinal models are being developed to study this organ in health and disease. Intestinal explants can be used in such investigations since they contain all the major intestinal cell types. A detailed procedure to isolate intestinal explants from the rat jejunum is described. A protocol for culturing them in vitro for up to 24 hr is also provided. © 2019 by John Wiley & Sons, Inc.
PMID: 31120607 [PubMed - as supplied by publisher] ...
Source: Current Protocols in Toxicology - May 25, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.
Authors: Kamalian L, Douglas O, Jolly CE, Snoeys J, Simic D, Monshouwer M, Williams DP, Park BK, Chadwick AE
Abstract
Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantage...
Source: Current Protocols in Toxicology - May 8, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Reliable Preclinical Model to Study the Impact of Cigarette Smoke in Development and Disease.
Authors: Aedo G, Miranda M, Chávez MN, Allende ML, Egaña JT
Abstract
The World Health Organization has estimated that, worldwide, cigarette smoking has caused more than 100 million deaths in the last century, a number that is expected to increase in the future. Understanding cigarette smoke toxicity is key for research and development of proper public health policies. The current challenge is to establish a reliable preclinical model to evaluate the effects of cigarette smoke. In this work, we describe a simple method that allows for quantifying the toxic effects of cigarette smoke using zebrafish. Here,...
Source: Current Protocols in Toxicology - May 8, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Toxicology in the Super-Resolution Era.
Authors: Cole R
Abstract
Light microscopy has played a central role in science for the past couple of hundred years and will continue to do so. Multiple super-resolution microscopy techniques have been in the headlines for smashing what for more than 100+ years was believed to be the limits of optical microscopy. This resolution improvement enables the visualization of molecular structures and processes on the nano scale. While certain scientific questions in toxicology can benefit from modalities within the super-resolution suite, due diligence is required for efficiency and to achieve optimal results. Fo...
Source: Current Protocols in Toxicology - April 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization.
Authors: Wheeler JH, Young CKJ, Young MJ
Abstract
A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside revers...
Source: Current Protocols in Toxicology - April 15, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Generation of Macrophages from Cynomolgus-Monkey Bone Marrow as a Model to Evaluate Effects of Drugs on Innate Immunity.
Authors: Li N, Ludmann SA, Anest L, He C, Narayanan PK
Abstract
Macrophages are innate immune cells that play important roles in various physiological and pathological processes. Evaluation of pro-inflammatory effects of drugs on macrophages has become commonplace in preclinical drug development prior to human clinical trials. Despite their body-wide distribution, tissue macrophages are often difficult to collect from large animals and humans in a noninvasive manner. Therefore, in vitro-differentiated macrophages are important tools to facilitate cross-species analysis of macrophage function. Although cyno...
Source: Current Protocols in Toxicology - April 15, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions.
This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perf...
Source: Current Protocols in Toxicology - April 7, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The Use of Intracerebral Microdialysis to Elucidate Environmentally Induced Neurotoxic Mechanisms.
This article provides an overview of the manner in which the methodology of intracerebral microdialysis is utilized in the field of neurotoxicology to elucidate the actions of environmental agents. The technique is employed in a variety of creative ways to address specific experimental goals involving myriad toxicants. With appropriate consideration of method parameters, investigators have also been able to address mechanistic issues in their studies. These investigations consist of sampling of neurotransmitters in extracellular fluid after various protocols of environmental metal exposure as well as assessments of blood-b...
Source: Current Protocols in Toxicology - April 4, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Isolation of Alveolar Type II Cells from Adult Bovine Lung.
Authors: Lee DF, Chambers MA
Abstract
Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often requ...
Source: Current Protocols in Toxicology - March 16, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Preparation and Measuring of Brain GABAA R-coupled Cl- /HCO3 - - Activity for Integral Assessment of Aquatic Toxicity.
In this study, we present a protocol for isolation of the membrane-bound Cl- /HCO3 - -ATPase from neuronal cells of animal brain. We then describe an uncomplicated in vitro method for measuring this ATPase activity for assessment of toxicity after interaction of this protein with an aquatic sample. This assay offers new avenues for using the Cl- /HCO3 - -ATPase as a biomarker of water toxicity. This biotest is efficient, requires very little of the enzyme, and retains its sensitivity at low levels of various compounds. © 2019 by John Wiley & Sons, Inc.
PMID: 30843667 [PubMed - as supplied by publisher] (Source...
Source: Current Protocols in Toxicology - March 9, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Inducing a Mucosal Barrier-Sparing Inflammatory Response in Laboratory-Grown Primary Human Nasal Epithelial Cells.
Authors: Ramezanpour M, Bolt H, Psaltis A, Wormald PJ, Vreugde S
Abstract
Here we use the toll-like receptor (TLR) 3 agonist poly I:C (LMW) to induce an inflammatory response in cells of submerged and/or air-liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). The inflammatory response is determined by measuring interleukin-6 (IL-6) protein levels by enzyme-linked immunosorbent assay (ELISA). The mucosal barrier integrity is determined by measuring transepithelial electrical resistance (TEER) and passage of fluorescently labeled dextrans. Stimulation with poly (I:C) LMW induces a 15- to...
Source: Current Protocols in Toxicology - February 6, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia.
Authors: Narayanan PK, Li N
Abstract
Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mono...
Source: Current Protocols in Toxicology - January 24, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Screening for Neurotoxicity with Microelectrode Array.
This article describes the preparation and use of a 48-well microelectrode array (MEA) platform along with custom data analysis algorithms and commercially available analysis tools to screen for neurotoxic liabilities and seizurogenic effects using recorded spike file data generated from cryogenically preserved rat cortical neurons. © 2018 by John Wiley & Sons, Inc.
PMID: 30575314 [PubMed - as supplied by publisher] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - December 25, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
