Current protocols in toxicology最新文献

Cover: In Santamaría et al. (https://doi.org/10.1002/cptx.89), the image shows (A) The whole ovary was serially sectioned (5 µm thick) and 1 out of every 10 sections was stained with picrosirius-hematoxylin (P-H) for morphological observation. Magnification: (B) 100×, (C) and (D) 400×. Red circles represent oocytes belonging to nests. Pink arrow represents primary follicle; blue arrow, early primary follicle; and green arrow, primordial follicle.

Protein-protein interactions are important in human disease. Developing and refining tools to understand physical contacts between signaling proteins is crucial. This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed. Step-by-step instructions are provided for performing these assays using the NS2B-NS3 interaction as an example. NS2B is an essential cofactor for flaviviral NS3 protease function. Advantages and disadvantages of these assays are further discussed. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Expression and purification of fusion proteins

Basic Protocol 2: Analysis of prey/bait pairs by SLC-based NS2B-NS3 interaction assay

Support Protocol 1: Interaction specificity assay

Support Protocol 2: Competition binding assay: Dose-response inhibition using cold prey or bait

Support Protocol 3: Competition binding assay: Inhibition by MBP-NS3 versus irrelevant MBP tag

Support Protocol 4: SLC-based NS2B-NS3 interaction assay using NS2B mutations known to disrupt NS2B-NS3 interactions

We describe a detailed protocol to establish a newborn rat whole ovary culture, which enables the study of direct effects (independent of hypothalamic-pituitary-gonadal axis) of endocrine disrupting chemicals (EDCs), such as benzophenone-3 (BP-3). This method is useful to understand changes in follicle formation, primordial to primary transition, and expression of regulatory molecules linked to these processes and also provides an alternative to animal models. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Rat ovarian surgery

Basic Protocol 2: Whole organ/ovarian culture

Basic Protocol 3: RNA isolation and quantitative real-time PCR

Basic Protocol 4: Histological processing and staining

In order to circumvent ethical, technical, and economic drawbacks regarding the use of animal serum in cell culturing, it is possible to adapt mammalian cells to serum-free media. Nowadays, there are several serum-free formulations available, including fully animal derived–free and chemically defined media, and different adaptation techniques. This article focuses on the gradual adaptation of a mammalian suspension cell culture to a chemically defined medium. The first step is to transfer the cells cultured in medium supplemented with fetal bovine serum (FBS) to a chemically defined medium of your choice, containing the same amount of FBS. The next steps consist of progressively reducing the amount of FBS, while monitoring cell growth and viability up to the complete elimination of FBS. This protocol has been successfully used to adapt THP-1 cells to a chemically defined medium with similar maximum specific growth rate as those cultured with FBS. © 2019 by John Wiley & Sons, Inc.

Basic Protocol: Gradual adaptation to chemically defined medium

Alternate Protocol: Direct adaptation to chemically defined medium

Support Protocol 1: Determining maximum specific growth rate of a cell culture

Support Protocol 2: Cell freezing and thawing

Herein, we describe a protocol for the preparation and analysis of primary isolated rat hepatocytes in a 3D cell culture format described as spheroids. The hepatocyte cells spontaneously self-aggregate into spheroids without the need for synthetic extracellular matrices or hydrogels. Primary rat hepatocytes (PRHs) are a readily available source of primary differentiated liver cells and therefore conserve many of the required liver-specific functional markers, and elicit the natural in vivo phenotype when compared with common hepatic cells lines. 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

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 fertilization and present a breeding strategy for obtaining tissue-specific knockouts. Finally, methods are provided for validating the knockout mice using PCR of genomic DNA, reverse-transcription PCR and quantitative reverse-transcription PCR of mRNA, and immunoblot analysis of proteins. © 2019 by John Wiley & Sons, Inc.

Cover: In Cole (https://doi.org/10.1002/cptx.77), the image shows ricin co-localization with the endoplasmic reticulum (ER). (A) LSCM ER is shown in blue and ricin co-localization is shown in yellow. (B) STED ER is shown in blue and LSCM ricin colocalization is shown in cyan. (C, D) 3-D enlargement of the area within the black box from panels (A) and (B). The co-localization of ricin with the ER is more accurate due to the improved resolution when imaging the ER with STED. Arrows in panels (C) and (D) correspond to ricin particles that are not co-localized with the ER. Scale bar: (B) 15 μm, (D) 10 μm for the zoomed-in image. Reprinted (adapted) with permission from Herrera et al. (2016). Copyright (2016) Cambridge University Press.

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.

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 advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc.

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, viability of larvae and adult fish, as well as the effects of cigarette smoke extracts on vascular development and tissue regeneration, can be easily assayed. © 2019 by John Wiley & Sons, Inc.