Current Protocols最新文献

This article describes procedures for infecting adult mice with murine cytomegalovirus (MCMV) and for infecting newborn mice to model congenital CMV infection. Methods are included for propagating MCMV in cell cultures and preparing a more virulent form of MCMV from the salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, methods are described for preparing the murine embryonic fibroblasts used for propagating MCMV, and for the plaque assay. © 2022 Wiley Periodicals LLC.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

The salivary glands produce saliva and are important in maintaining oral health. Saliva keeps the mouth moist, cleanses the oral cavity, aids digestion, and has antibacterial properties. Saliva also helps in swallowing and speech. Investigating the development of the salivary glands is thus relevant in the context of both health and disease. Various cell culture methods have been used to study salivary gland development, including culturing cells in two dimensions (2D). Under physiological conditions, cells constantly interact with other cells and the extracellular matrix, which controls complex biological functions such as cell migration and apoptosis, and can modulate gene expression. Since many of these functions are not accurately represented or reproduced in 2D culture, the results of in vitro experiments using such culture methods are often not reflected in vivo. The use of 3D cultures, such as organ cultures, has helped address this issue and has emerged as a model that better reflects the in vivo physiological environment. Here, we describe a protocol for establishing submandibular salivary gland organ culture that is more concise and simpler than previous methods and includes the separation and dissection of the salivary glands. We also describe the use of environmental stress (hypoxic stimulation) and inhibitors (U0126, LY294002, and rapamycin) to elucidate signaling pathways involved in salivary gland development. This protocol can provide researchers with a simpler and more robust method of salivary gland organ culture, enabling analysis of organ-based signaling pathways to advance developmental biology research. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Submandibular salivary gland organ culture Basic Protocol 2: Analysis of salivary gland development in the presence of hypoxia and signaling pathway inhibitors Basic Protocol 3: Western blotting using submandibular salivary gland organ culture.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells known to play a role in perpetuating a wide range of pathologies, such as chronic infections, autoimmune diseases, and cancer. MDSCs were first identified in mice by the markers CD11b⁺ Gr1⁺ , and later, based on their morphology, they were classified into two subsets: polymorphonuclear MDSCs, identified by the markers CD11b⁺ Ly6G⁺ Ly6C^Low , and monocytic MDSCs, detected as being CD11b⁺ Ly6G^- Ly6C^Hi . MDSCs are studied as immunosuppressive cells in various diseases characterized by chronic inflammation and are associated with disease causes/triggers such as pathogens, autoantigens, and cancer. Therefore, different diseases may diversely affect MDSC metabolism, migration, and differentiation, thus influencing the generated MDSC functional features and ensuing suppressive environment. In order to study MDSCs in a pathology-free environment, we established and calibrated a highly reproducible mouse model that results in the development of chronic inflammation, which is the major cause of MDSC accumulation and immune suppression. The model presented can be used to study MDSC phenotypes, functional diversity, and plasticity. It also permits study of MDSC migration from the bone marrow to peripheral lymphatic and non-lymphatic organs and MDSC crosstalk with extrinsic factors, both in vivo and ex vivo. Furthermore, this model can serve as a platform to assess the effects of anti-MDSC modalities. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Repetitive M.tb immunizations for the induction of chronic inflammation Alternate Protocol 1: Creating a lower grade of inflammation by changing the site of immunization Alternate Protocol 2: In vivo evaluation of immune status Support Protocol 1: Preparation of reconstituted M.tb aliquots and M.tb-IFA emulsions for each of the three injections Support Protocol 2: Preparation of an ovalbumin lentiviral expression vector Support Protocol 3: Fluorescence titering assay for the lentiviral expression vector Support Protocol 4: Spleen excision, tissue dissociation, and preparation of a single-cell suspension Support Protocol 5: Labeling of splenocytes with CFSE proliferation dye.

The intentional pharmacological manipulation of myogenesis is an important technique for understanding the underlying mechanisms of muscle differentiation and disease etiology. Using the pharmacological agent metformin as an example molecule, we present a systematic approach to examine the impact of pharmacological agents on the myogenic program. This consists of optimizing the in vitro differentiation of primary myoblast cells followed by the generation of a dose-response curve for a respective pharmaceutical. To assess myogenic differentiation, we utilized three approaches (incorporating both transcriptional and protein techniques) to assess the effects of biologically active agents on the in vitro differentiation of primary myogenic progenitors. First, the immunofluorescent visualization of myosin heavy chain (MYHC), which is expressed in differentiated myofibers, is used to obtain the fusion index, a quantitative read-out of differentiation efficiency. Second, quantitative reverse transcription PCR (RT-qPCR) reveals the expression of myogenic factors (Pax7, Myf5, Myod, Myog, Myh2) at the transcript level. Third, western blotting is used to assess the protein expression levels of the myogenic markers (PAX7, MYF5, MYOD, MYOG, and MYHC). By monitoring the expression of these various myogenic factors during the differentiation process, the relative cellular state and differentiation status between samples can be determined. Combined, these approaches enable the successful assessment of the impact of pharmacological agents on myogenic differentiation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Immunofluorescence assay for qualitative and quantitative assessment of pharmacological agents on in vitro myogenic differentiation Support Protocol 1: Evaluating myogenic gene expression by RT-qPCR Support Protocol 2: Evaluating myogenic protein expression by western blot.

In situ hybridization has been a robust method for detection of mRNA expression in whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in situ hybridization have incorporated oligo-based probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is third-generation in situ hybridization chain reaction (V3HCR). Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryosectioning, tissue fixation, and processing over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly autofluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin, we determined that application of a lipofuscin quencher dye is compatible with V3HCR, in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in situ detection of gene expression in human neuronal populations that are otherwise problematic due to lipofuscin autofluorescence. © 2022 Wiley Periodicals LLC. Basic Protocol: Mouse and human fresh-frozen tissue in situ hybridization chain reaction on microscope slides Support Protocol: Aliquoting of HCR probes and hairpins.

Different populations of immune cells rely on their distinct migration patterns for immunosurveillance, immune regulation, tissue specific differentiation, and maturation. It is often important to clarify whether cells are recirculating or tissue resident, or whether tissue-specific cells are derived from blood-borne precursors or a tissue-resident population. Though migration or tissue residency of immune cells critically depends on the expression of different homing molecules (chemokine receptors, tissue retention molecules, etc.), characterization based solely on the expression of homing molecules may not faithfully reflect the migration patterns of immune cells. Therefore, a more reliable method to clarify migration patterns of immune cells is required. Parabiosis is a surgical connection of two mice resulting in a shared circulatory system, which allows reliable distinction of tissue-resident and circulating cells. Here, we describe a set of protocols for parabiosis, including technique details, pitfalls, and suggestions for optimization and troubleshooting. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of mice for parabiosis surgery Basic Protocol 2: Parabiosis surgery Basic Protocol 3: Recovery and use of mice after parabiosis surgery Basic Protocol 4: Reversal of parabiotic surgery Basic Protocol 5: Analysis of parabionts.

The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.