Methods in molecular biology
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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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"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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Animal models of traumatic brain injury (TBI) provide important tools for studying the pathobiology of brain trauma and for evaluating therapeutic or diagnostic targets. Incorporation of additional insults such as hemorrhagic shock (HS) and/or hypoxemia (HX) into these models more closely recreates clinical scenarios as TBI often occurs in conjunction with these systemic insults (i.e., polytrauma). ⋯ The physiological, histological, and behavioral aspects of this animal model have been characterized and have demonstrated exacerbating effects of systemic insults on penetrating TBI. As such, this model may facilitate the use of simultaneous assessments of multiple mechanisms and provide a platform for testing novel diagnostic and therapeutic targets.
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Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.
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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.