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 platform relies on a high-speed solenoid device to mimic explosive injury scenarios, allowing for large, complex injury shapes and sizes that are often observed in casualties and are more difficult to treat. The system can be implemented with precise computer control of the injury mechanism to allow for more complex setups. Also, the system can make use of real-time intraocular pressure measurement to track changes during injury induction and to assess therapeutic efficacy for restoring intraocular pressure and the integrity of the eye. These protocols will assist with implementation of the injury model in prospective laboratories seeking to develop therapeutics or studying biological changes that occur from this type of traumatic injury. Published 2020. U.S. Government. Basic Protocol 1: Preparing gelatin molds and porcine eye tissue Basic Protocol 2: Creating an open-globe injury using a solenoid device Alternate Protocol 1: Constructing a computer-controlled system for open-globe injury Alternate Protocol 2: Constructing a pressure measurement system for tracking intraocular pressure Support Protocol 1: Assessing ocular compliance in porcine eyes Support Protocol 2: Assessing outflow rate from the anterior chamber Support Protocol 3: Assessing burst pressure in porcine eyes.

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 improvements, which have been aided by advancements made in flow cytometry technology and the development of fluorescent reagents. The protocols presented here describe flow cytometry-based techniques for PBMC culture that can be employed to determine the impact of various environmental toxicants on the immune system. A major advantage of these procedures is that they will provide information about a toxicant or drug through in vitro methods. As the relationship between exposures to certain toxicants and an individual's response to vaccinations has been of concern, one of the protocols described shows how to test an environmental toxicant for potential modification of the immune response to vaccine antigen(s). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of cell proliferation Support Protocol 1: Blood cell counting Support Protocol 2: Measurement of cell viability after culturing Basic Protocol 2: Identification of affected naïve/memory cell subsets.

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.

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.

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. For a given hypothesis being tested, there are biophysical issues that need to be considered before heading down the super-resolution road. All commercially available super-resolution modalities, along with cautions and tips, will be discussed. © 2019 by John Wiley & Sons, Inc.

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 17-fold increase in IL-6 production by HNEC-ALI cells. © 2019 by John Wiley & Sons, Inc.

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 mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley & Sons, Inc.

Neurotoxicity and seizurogenic liabilities are difficult to detect using currently available in vitro cytotoxicity assays. This is primarily due to the inherent limitations of these assays to predict adverse neural network disruptions and chemically induced perturbations. Many of these detrimental effects are detected with in vivo studies after substantial time and monetary resources have already been invested. Due to these late-stage unforeseen side effects, the implementation of a reliable high throughput in vitro method for assessing seizure-inducing and neurotoxic compound effects early in the drug discovery process would be ideal. We have developed an in vitro screening tool to identify chemical entities that cause neurotoxic and seizurogenic effects. 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.

In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three-dimensional primary cells containing both primary epithelial and mesenchymal components from mouse gastrointestinal tissues. ALI organoids accurately recapitulate organ structures, multilineage differentiation, and physiology. Since ALI organoids from human tissues have not been produced, we modified the previous protocol for mouse ALI organoid culture to establish the culture system of ALI organoids from normal and tumor colorectal tissues of human patients. The current unit presents a protocol for preparation of the ALI organoid culture from normal and tumor colorectal tissues of human patients. ALI organoid culture from human tissues might be useful for examining not only resistance to chemotherapy in a tumor microenvironment but also toxic effects on organoids. © 2018 by John Wiley & Sons, Inc.

Cannabinoid compounds refer to a group of more than 60 plant-derived compounds in Cannabis sativa, more commonly known as marijuana. Exposure to marijuana and cannabinoid compounds has been increasing due to increased societal acceptance for both recreational and possible medical use. Cannabinoid compounds suppress immune function, and while this could compromise one's ability to fight infections, immune suppression is the desired effect for therapies for autoimmune diseases. It is critical, therefore, to understand the effects and mechanisms by which cannabinoid compounds alter immune function, especially immune responses induced in autoimmune disease. Therefore, this unit will describe induction and assessment of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and its potential alteration by cannabinoid compounds. The unit includes three approaches to induce EAE, two of which provide correlations to two forms of MS, and the third specifically addresses the role of autoreactive Tcells in EAE. © 2018 by John Wiley & Sons, Inc.