Methods in molecular biology
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"pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.
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Immune checkpoint inhibitors (ICI) are a new class of drugs characterized by their ability to enhance antitumor immune responses through the blockade of critical cell surface receptors involved in the maintenance of peripheral tolerance. The recent approval of ICI targeting CTLA-4 or PD-1 for the treatment of cancer constitutes a major breakthrough in the field of oncology and demonstrates the potential of immune-mediated therapies in achieving durable cancer remissions. The identification of new immune regulatory pathways that could be targeted to reactivate or boost antitumor immunity is now a very active field of research. ⋯ In this chapter, we describe the general methodology to evaluate antitumor activity of ICI in immunocompetent mice. We outline protocols to reliably establish tumors in mice and generate lung metastasis through tail vein injections with the aim of testing the efficacy of ICI. We also present methods to analyze the composition of the tumor immune-infiltrate by multicolor flow cytometry.
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In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Animal models provide unmitigated access to histological samples, which provide a "direct measure" of damage. However, even with unconstrained access to tissue morphology and biochemistry assays, the findings typically do not account for loss of muscle function. ⋯ The majority of animal models testing contractile force have been limited to the muscle groups moving the ankle, with advantages and disadvantages depending on the equipment. Here, we describe in vivo methods to measure torque, to produce a reliable muscle injury, and to follow muscle function within the same animal over time. We also describe in vivo methods to measure tension in the leg and thigh muscles.
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The impact acceleration (I/A) model of traumatic brain injury (TBI) was developed to reliably induce diffuse traumatic axonal injury in rats in the absence of skull fractures and parenchymal focal lesions. This model replicates a pathophysiology that is commonly observed in humans with diffuse axonal injury (DAI) caused by acceleration-deceleration forces. Such injuries are typical consequences of motor vehicle accidents and falls, which do not necessarily require a direct impact to the closed skull. ⋯ Furthermore, the trauma device is inexpensive and readily manufactured in any laboratory, and the induction of injury is rapid (~45 min per animal from weighing to post-injury recovery) allowing multiple animal experiments per day. In this chapter, we describe in detail the methodology and materials required to produce the rat model of I/A in the laboratory. We also review current adaptations to the model to alter injury severity, discuss frequent complications and technical issues encountered using this model, and provide recommendations to ensure technically sound injury induction.
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Physical trauma in the central nervous system (CNS) is usually the result of a number of forces in different directions and dimensions. A large number of experimental models have been developed to improve the possibilities to understand the outcome of CNS trauma. ⋯ Models can serve different needs, such as: to test new treatments for injuries, to reveal thresholds for injuries, to provide a better understanding of injury mechanisms, or to test tools and methods for translation between experiments and clinical data. In this chapter, we will discuss on the validation of models and translation between experimental and clinical studies.