Phenotypic and Functional Evaluation of Hematopoietic Stem and Progenitor Cells in Toxicology of Heavy Metals.
Authors: Li Q, Yang Z, Zhao Y, Jia X, Zhou Z, Zhang Y
Abstract
All blood cells are differentiated from hematopoietic stem and progenitor cells (HSPCs), a process known as hematopoiesis that can be influenced by a variety of environmental factors. In this unit, we introduce a couple of protocols including phenotypic analysis, mixed bone marrow (BM) chimera and rescue assays, colony-forming unit (CFU), and in vitro OP9 stromal cell differentiation assays for evaluating HSPCs in the BM of mice, and provide some examples of their implications in mouse models treated with heavy metals. The protocols for evaluat...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Preparation of Human Primary Colon Tissue-Derived Organoid Using Air Liquid Interface Culture.
Authors: Usui T, Sakurai M, Umata K, Yamawaki H, Ohama T, Sato K
Abstract
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 produc...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Development of an In Vitro Method to Estimate the Sensitization Induction Level of Contact Allergens.
Authors: Galbiati V, Gibbs S, Roggen E, Corsini E
Abstract
Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens, based on the use of Reconstituted human Epidermis (RhE).© 2018 b...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Evaluation of Marijuana Compounds on Neuroimmune Endpoints in Experimental Autoimmune Encephalomyelitis.
Authors: Kaplan BLF
Abstract
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 alte...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Real-Time Image-Based Approach to Distinguish and Discriminate Apoptosis from Necrosis.
Authors: Lekshmi A, Varadarajan SN, Lupitha SS, Nair M, Chandrasekharan A, Santhoshkumar TR
Abstract
Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing sti...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).
Authors: Garg U, Munar A, Frazee C
Abstract
Mycophenolic acid (MPA) is an immunosuppressant that is used in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring of MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, due to their higher specificity, mass spectrometry methods are preferred. In this unit, we describe a liquid chromatography tandem mass spectrometry (LC/MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of levels of...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Use of Human Embryoid Bodies for Teratology.
Authors: Flamier A, Singh S, Rasmussen TP
Abstract
Human birth defects are relatively common and can be caused by exposure to environmental teratogens or to pharmaceuticals with teratogenic activities. Human embryonic stem cells (hESCs), by virtue of their pluripotent nature, provide an excellent cellular platform for teratogen detection and risk assessment. This unit describes detailed protocols for the preparation and validation of highly pluripotent hESCs, the production of large quantities of aggregated multicellular spheroids composed of hESCs, and these spheroids' differentiation into embryoid bodies...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Gas Chromatography Mass Spectrometry (GC-MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine.
Authors: Ketha H, Webb M, Clayton L, Li S
Abstract
Phenethylamine derivatives are being increasingly exploited for recreational use as "designer" stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4-methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design phenethylamines so the user can bypass legal action when detected, as many of these are yet to be regulated by government authorities. Substituted phenethylamines or 2C amines, N-methoxybenzyl derivatives of the corresponding 2C amines commonly known as NBOMe compound...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Inter-and Intra-Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation.
Authors: Gupta S, Sharma R, Agarwal A
Abstract
The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation. Molecular anomalies in the sperm can be detected by a sperm DNA fragmentation (SDF) assay which can be used in adjunct to conventional semen analysis. While the sperm chromatin structure assay (SCSA) remains the "gold standard," the TUNEL assay using flow cytometry is be...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Co-Culture of Neurons and Microglia.
Authors: Roqué PJ, Costa LG
Abstract
Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro- and anti-inflammatory cytokines and reactive oxygen species, that contribute to modulating neuro-inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, ...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Overview of Neurotoxicology.
Authors: Costa LG
Abstract
The nervous system has a central and primary function in the body, and its relevance and complexity make it a target for a large number of toxic substances. The most common forms of neurotoxicity are the death of neurons (neuronopathy), the degeneration of axons (axonopathy), damage to glial cells (e.g., myelinopathy), and interference with the axonal membrane or neurotransmission. Important neurotoxicants are found among pesticides, metals, solvents, natural substances, and industrial chemicals. Environmental chemicals may also contribute to the etiopathogenesis of neurodevelopm...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells.
Authors: Lauer FT, Denson JL, Burchiel SW
Abstract
This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and phenotype stability are not altered through cryopreservation and extended storage. For this analysis, HPBMC were isolated from 7 healthy individuals, and 11-color flow cytometry was performed on freshly isolated samples as well as samples cryopreserved for short- an...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring β-Galactosidase Activity in Gram-Positive Bacteria Using a Whole-Cell Assay with MUG as a Fluorescent Reporter.
Authors: Chiu NHL, Watson AL
Abstract
The use of β-galactosidase enzyme as a biomarker has the potential to determine activity levels of the microbiome of a variety of organisms due to its common presence in both eukaryotes and prokaryotes. Completing the assay in a whole-cell format facilitates the monitoring of β-galactosidase activity in its actual cellular environment. This unit describes an optimized fluorescent assay for β-galactosidase that has enough sensitivity to detect the enzymatic activity despite the thick gram-positive bacterial cellular membrane. The use of a smaller fluorometric substra...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Metallic Nano-Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers.
Authors: Pecoraro R, Salvaggio A, Marino F, Caro GD, Capparucci F, Lombardo BM, Messina G, Scalisi EM, Tummino M, Loreto F, D'Amante G, Avola R, Tibullo D, Brundo MV
Abstract
Nanomaterials (NM) have different shapes and can be composed of different materials such as carbon, silicon, and some metals like gold, silver, and titanium. They are used as fillers, catalysts, semiconductors, cosmetics, drug carriers in medicine, energy storage systems, and antifriction coatings. NM are the parent compounds of nanoparticles (NPs), which may be divided into two groups: fullerenes and engineered nanoparticles (ENPs). ...
Source: Current Protocols in Toxicology - November 9, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety.
Authors: Al-Yousuf K, Webster CA, Wheeler GN, Bombelli FB, Sherwood V
Abstract
The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use ...
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
