Methods in cell biology最新文献

Basophils constitute a rare population of granulocytes with key functions in allergies, immunodeficiencies and cancer. The scarcity of basophils in human blood and tissues constitutes a considerable limit for the study of these cells. Interleukin-3 (IL-3) stimulates both the differentiation and the expansion of basophils from bone marrow (BM) precursors by positively regulating the expression of the IL3Ra receptor. We have standardized an in vitro differentiation protocol of mouse basophils (mBaso) from BM precursors through culture in presence of IL-3 for 10 days followed by cell sorting. At the end of the 10-day differentiation, a considerable number of mBaso can be obtained and cell sorting procedures further improved the isolation of an extraordinarily pure (>98 %) and vital FcεR1⁺ CD11c^- c-kit^- mBaso population. Phenotypic analysis of terminally differentiated (day 10) unsorted mBaso cultured for 24 h showed a decrease in basophilic lineage (c-kit^-) and an increase of mastocytic lineage (c-kit⁺) and reduced the expression of basophil markers FcεRI, CD49b and CD200R either in absence of stimuli or following activation with the alarmin IL-33, indicating cell dedifferentiation. In contrast, terminally differentiated and FcεR1⁺ CD11c^- c-kit^- sorted mBaso do not dedifferentiate in mast cells when placed in culture, and responded to IL-33 stimulation by up-regulating the activation marker CD63 without down-modulation of FcεRI and CD200R3. These evidences highlight that in vitro differentiation followed by cell sorting is a useful method to obtain elevated numbers of highly pure mBaso that preserve their lineage markers and thus are suitable for conducting the desired functional studies.

Mesentery is a crucial part of an animal's digestive system since it holds the intestine in place, while also contains the specialized lymph nodes and immune cells that help protect the intestines from infections and support the body's immune response in the abdominal cavity. Analyzing mesenteric lymph can help better understand the transport mechanisms and potential implications for various conditions, such as lymphatic disorders or underlying infections. The first step towards this is the precise collection of the mesenteric lymph. In this chapter, we describe the optimized protocol of cannulation to collect afferent and efferent mesenteric lymphatic fluid.

Mitochondrial dysfunction is a shared hallmark of neurodegenerative disorders, including Alzheimer's disease (AD) and tauopathies among others. Pathological alterations of the microtubule-associated protein Tau can disrupt mitochondrial dynamics, transport, and function, ultimately leading to neuronal toxicity and synaptic deficits. Understanding these processes is crucial for developing therapeutic interventions. The nematode Caenorhabditis elegans serves as a powerful model to study mitochondrial morphology and Tau-induced neurotoxicity due to its well-characterized nervous system and genetic tractability. Here, we describe a robust methodology for assessing mitochondrial morphology, Tau aggregation, and neuronal integrity in a nematode model of tauopathy. By combining confocal laser scanning microscopy and motility assays, we provide a comprehensive framework for investigating mitochondrial deficits. This approach offers valuable insights into the interplay between Tau pathology and mitochondrial dysfunction, thereby advancing our understanding of neurodegenerative mechanisms and potential therapeutic targets.

This chapter introduces to the indications, biofluids used and laboratory methods of liquid biopsy. A detailed description of preanalytical factors, extraction methods, enrichment methods, quality control, storage and analysis of various targets of liquid biopsy such as Circulating Tumor Nucleic Acids, Circulating Tumor Cells, Extracellular Vesicles, and Tumor-educated platelets has been included.

Cancer is perceived as difficult to treat due to its capacity to manifest in different forms and lack of knowledge of mechanical details. Advances in the analytical techniques relevant to boosting cancer research require an hour. With the help of cell culture techniques, several significant advances in cancer research have been made in the recent past. The main difficulty associated with cell culture techniques of cancer research is to create an in vivo tumor microenvironment cost-effectively. Here, in this chapter, we have discussed the different protocols for utilizing cell culture techniques in cancer research. The 2D, 3D, scaffold and organoid based cell culture techniques have been covered in detail. In addition, we have presented a comparative analysis, including advantages and disadvantages of each type of cell culture technique. Moreover, the assays, which can be used for assessing the quality of cancer cell lines, have been listed in detail.

The externalization of Phosphatidylserine (PS) from the inner surface of the plasma membrane to the outer surface of the plasma membrane is an emblematic event during apoptosis and serves as a potent "eat-me" signal for the efferocytosis of apoptotic cells. Although less well understood, PS is also externalized on live cells in the tumor microenvironment and on live virus-infected cells whereby it serves as an immune modulatory signal that drives tolerance and immune escape. Given the importance of PS in cancer immunology and immune escape, PS-targeting monoclonal antibodies have been characterized with promising immunotherapeutic potential. Here, we describe the cloning and characterization of a series of PS targeting antibodies and their potential use and utility in immuno-oncology.

Neutrophils are pivotal in orchestrating tumor-induced systemic inflammation and are increasingly recognized for their critical involvement in both the initiation and progression of cancer. A fundamental facet of neutrophil biology is their migratory capacity, which enables them to extravasate and infiltrate tumors in other tissues, where they carry out essential effector functions. Unraveling the intricate mechanisms of neutrophil motility and migration is crucial for comprehending immune responses and inflammatory processes, shedding light on their substantial contribution to cancer progression. Here, we provide a comprehensive protocol to assess direct ex vivo motility and migration of freshly isolated human neutrophils, offering valuable insights into their behavior.

The Innate Lymphoid Cells (ILCs) are a family of innate immune cells composed by the Natural Killer (NK) cells and the helper ILCs (hILCs) (ILC1, ILC2, ILC3), both developing from a common ILC precursor (ILCP) derived from hematopoietic stem cells (HSCs). A correct ILC reconstitution is crucial, particularly in patients receiving HSC transplantation (HSCT), the only therapeutic option for many adult and pediatric high-risk hematological malignancies. Indeed, mainly thanks to their cytotoxic activity, NK cells have a strong Graft-versus-Leukemia (GvL) effect. On the other hand, hILCs, that are mainly tissue resident, are involved in tissue repair and homeostasis, Graft-versus-Host Disease (GvHD) prevention and immune response to infections. Unlike NK cell development, hILC-poiesis is still poorly characterized in humans. Here, we provide a protocol for the in vitro ILC differentiation from healthy donor peripheral blood-derived CD34⁺ HSCs. This could represent a useful model to dissect the molecular mechanisms by which the distinct ILC subsets are generated from ILCP leading to the development of novel strategies to improve the HSCT clinical outcome.

Treatment with autologous chimeric antigen receptor (CAR)-modified T cells can achieve outstanding clinical response rates in heavily pretreated patients with B and plasma cell malignancies. However, relapses occur, and they limit the efficacy of this promising treatment approach. The complex GMP-compliant production and high treatment costs cause that CAR T cells cannot yet be used in a broad population. Among others, CAR T cell therapy has evolved regarding vector design and manufacturing process. Optimal production of CAR T cells is not yet defined, far from being standardized. Quality, cellular composition and immunophenotype of the administered CAR T cells are influenced by the manufacturing protocol and therefore play a crucial role for therapeutic success. For the gene transfer, viral and non-viral strategies are available. Retrovirus-based protocols for CAR T cell production offer advantages in terms of stable gene integration, sufficient transduction efficiency, proven clinical success, and scalability. Here, we detail a retrovirus-based generation protocol of human CAR-modified T cells for experimental immunotherapeutic treatment of cancer cells. For the CAR generation, HEK-293-based packaging cell lines, CD3⁺ selection, CD3/CD28-coated bead-based activation and IL-2/IL-15-mediated expansion were used. This protocol can be applied for every possible CAR construct after being successfully transfected in HEK-293-based packaging cell lines.

In recent years, three-dimensional (3D) cultures of tumor cells has emerged as an important tool in cancer research. The significance of 3D cultures, such as tumor spheroids, lies in their ability to mimic the in vivo tumor microenvironment more precisely, offering a nuanced understanding of immune responses within the context of tumor progression. In fact, the infiltration of cytotoxic lymphocytes is key to determining patients' prognosis in several types of cancer and response to immunotherapy. Therefore, harnessing the cytotoxic and infiltration potential of immune cells is a promising avenue for developing effective therapies. This protocol offers a straightforward approach for analyzing infiltrating lymphocytes in tumor spheroids by confocal microscopy imaging. Although it specifically involves utilizing tumor spheroids and culture with autologous tumor-infiltrating T lymphocytes (TILs), the protocol can be adapted for other immune cell types, such as NK cells.