Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.
Authors: Hamze JG, Jiménez-Movilla M, Romar R
Abstract
We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction...
Source: Current Protocols in Toxicology - December 19, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction.
We describe how to use circumcision tissue obtained from the clinic to isolate FSK cells by mechanical and enzymatic disaggregation, how to use a cationic dye, crystal violet, which is retained by proliferating cells, to determine cell viability, and how to prepare samples to assess the metabolic status of cells by evaluating different mitochondrial parameters with transmission electron microscopy. We have successfully used the approaches outlined here to recapitulate physiological conditions in these cells in order to study the effects of increased intracellular levels of formaldehyde. © 2020 U.S. Government. Basic Proto...
Source: Current Protocols in Toxicology - November 19, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
An Open-Globe Porcine Injury Platform for Assessing Therapeutics and Characterizing Biological Effects.
Authors: Snider EJ, Edsall PR, Cornell LE, Gross BM, Butler JJ, Zawacki M, Boice EN
Abstract
Open-globe injuries can result in permanent vision loss, partly due to extended delays between injury and medical intervention. Even with early intervention, the management of open-globe injuries remains a challenge for ophthalmologists, mostly due to inadequate or suboptimal current therapies. To aid in the development of novel therapeutics and track toxicological and pathophysiological changes, this article details an open-globe injury platform capable of inducing injuries in enucleated porcine eyes. The injury p...
Source: Current Protocols in Toxicology - October 28, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.
Authors: Assis-de-Lemos G, Ledur PF, Karmirian K, Rehen SK, Galina A
Abstract
Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfu...
Source: Current Protocols in Toxicology - September 5, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET.
Authors: Dikovskaya D, Dinkova-Kostova AT
Abstract
The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-mediated stress response is a major cellular defense mechanism against endogenous and exogenous oxidants, electrophiles, and pro-inflammatory agents. A number of Nrf2 inducers are being developed to therapeutically stimulate this pathway. Inducers are typically sensed by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator and a binding partner of Nrf2. Modifications of Keap1 by oxidants or electrophiles, or its targeting by compounds that disrupt its interaction with Nrf2, alter the c...
Source: Current Protocols in Toxicology - August 15, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In Vitro Evaluation of Toxicant Influences on the Immune System.
Authors: Kasten-Jolly J, Lawrence DA
Abstract
Culture of human peripheral blood mononuclear cells (PBMCs) still remains a convenient and sensitive method for measurement of a person's immune system health. Basic elements of the process, namely PBMC purification and culture medium formulation, were first reported in the late 1960s, and the utility of the method for clinical application was reported in the 1970s. Clinically, the approach can provide information about the ability of an individual's immune system to fight off attacks by various pathogens. Over the years, the method has undergone many improveme...
Source: Current Protocols in Toxicology - June 17, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of Busulfan in Plasma by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Authors: Pablo A, Breaud AR, Clarke W
Abstract
Bone marrow transplantation is used to treat particular types of cancers such as lymphoma, leukemia, and multiple myeloma. Appropriate dosing of busulfan during the preparative phase is critical for a successful allograft; if blood concentrations get too high significant liver toxicity can occur, if blood concentrations are too low, then graft-versus-host disease (GVHD) can develop. Busulfan monitoring in blood allows hospitals with the opportunity to provide individualized medicine to patients and improve overall patient outcome. Liquid Chromatography-Tandem ...
Source: Current Protocols in Toxicology - May 30, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of Immunosuppressant Drugs in Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
Authors: Pablo AH, Breaud AR, Clarke W
Abstract
Immunosuppressant medications help suppress the immune system response through inhibition of various checkpoints in the regulatory biochemical pathway. This is useful in prevention of organ rejection in transplantation or in the treatment of autoimmune diseases such as lupus or rheumatoid arthritis. Quantification of immunosuppressive drugs in blood is needed clinically for optimization of treatment and to avoid toxicity or unwanted side effects. Here, we describe a quantitative method to determine the concentration of cyclosprine A, tacrolimus, sirolimus, an...
Source: Current Protocols in Toxicology - May 23, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Generating Bacterial Foods in Toxicology Studies with Caenorhabditis elegans.
Authors: Ke T, Santamaría A, Tinkov AA, Bornhorst J, Aschner M
Abstract
Caenorhabditis elegans is a free-living animal that is used as a powerful experimental model in biological sciences. The natural habitat of the animal are areas rich in material from rotting plants or fruits being decomposed by a growing number of microorganisms. The ecology of the natural habitat of C. elegans is a complex interactive network involving many species, including numerous types of bacteria, viruses, fungi, slugs, snails, and isopods, among which bacteria play multifaceted roles in the natural history of C. elegans. Under...
Source: Current Protocols in Toxicology - May 23, 2020 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Functional Assay to Assess Toxicity During Murine B Cell Development In Vitro.
Authors: Guilbert C, Chou H, Bolt AM, Wu TH, Luo VM, Orthwein A, Mann KK
Abstract
B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface...
Source: Current Protocols in Toxicology - December 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of Protein-Protein Interactions by Split Luciferase Complementation Assay.
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 ...
Source: Current Protocols in Toxicology - December 6, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Culturing Rat Whole Ovary for UV Filter Benzophenone-3 Treatment.
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 quanti...
Source: Current Protocols in Toxicology - November 26, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Adaptation of Mammalian Cells to Chemically Defined Media.
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...
Source: Current Protocols in Toxicology - November 24, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of Sertoli Cell Proliferation by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide and Sulforhodamine B Assays.
Authors: Martins AD, Oliveira PF, Alves MG
Abstract
The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short ...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assembly and Application of a Three-Dimensional Human Corneal Tissue Model.
Authors: McKay TB, Ford A, Wang S, Cairns DM, Parker RN, Deardorff PM, Ghezzi CE, Kaplan DL
Abstract
The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict ...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
