Methods in molecular biology最新文献

The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.

Adipose tissue is recognized not only as an endocrine organ but also as a reservoir for adipose-derived stromal/stem cells (ASCs). ASCs have stimulated the interest of both the scientific and medical communities due to their therapeutic potential and applications in tissue engineering and regenerative medicine. ASCs are leveraged for their multipotency and their paracrine function. ASC behavior is highly variable and donor dependent. Donor age, body mass index, disease status, sex, and ethnicity can lead to differential overall function and quality. The impact of donor age and passage on ASC behavior has been well documented, impacting cell proliferation and differentiation potential and thus must be taken into careful consideration when conducting in vitro studies. Pooling of ASCs from different donors reduces heterogeneity among individual donors and produces ASCs with a consistent differentiation and paracrine profiles, an advantage for studies in biological aging. This chapter provides a detailed overview for studies related to quality control for ASC pools considering biological and chronological aging in ASCs. There are hallmarks of biological aging and specific assays associated with the evaluation of each hallmark. Nevertheless, here we present the assays that provide a standardized characterization and qualification of donor pools for their regenerative potential, considering chronological and biological age of the pool. The assays included in this chapter are considered quality control standards to evaluate cell proliferation, differentiation, colony-forming units, and cellular senescence from different donor age and cell passage cohorts.

Regenerative medicine investigates the conversion of pancreatic ductal cells into functional islet cells, offering innovative treatments for conditions such as diabetes. Ductal cells, primarily supporting the pancreas' exocrine functions, can differentiate into various cell types, including islet cells, under specific conditions, opening new avenues in research and therapy. The outlined protocol elaborates on the conversion process, covering ductal cell differentiation induction, and insulin-producing capacity assessment. The primary objective is to address the shortage of insulin-secreting cells for transplantation, thereby advancing diabetes treatment methodologies.

The ex vivo myofiber culture system has proven to be a useful methodology to explore the biology and behavior of satellite cells within their niche environment. However, a limitation of this system is that myofibers and their associated satellite cells are commonly examined using conventional fluorescence microscopy, which renders a three-dimensional system into two-dimensional imaging, leading to the loss of precious information or misleading interpretation of observations. Here, we report on the use of light-sheet fluorescence microscopy to generate three-dimensional and live imaging of satellite cells on myofibers. Light-sheet microscopy offers high imaging speed and good spatial resolution with minimal photo-bleaching, allowing live imaging and three-dimensional acquisition of skeletal muscle fiber specimen. The potentials of this technology are wide, ranging from the visualization of satellite cell behavior such as cell division and cell migration to imaging the sub-cellular localization of proteins or organelles.

Autophagy refers to the natural cellular process by which cells degrade and recycle their own damaged or dysfunctional cellular components. It is an essential mechanism for maintaining cellular homeostasis removing toxic substances and providing energy during times of stress or nutrient deprivation. When autophagy is dysregulated or impaired, it can have detrimental effects on cell function and overall health. Studying autophagy in skin exposed to pollutants can provide valuable insights into the cellular mechanisms underlying pollutant-induced skin damage. Proteomic methods, which involve the large-scale analysis of proteins, can be employed to investigate the changes in protein expression associated with biological processes including autophagy. Here, we thus describe a method where LC-MS/MS was applied to identify the deregulated proteins in pollutant exposed-skin. Using bioinformatics and statistical analysis, we extracted the qualitative and quantitative information for proteins involved in autophagy. These deregulated proteins were then validated by immunohistochemistry (IHC). These methods help to understand how the pollutants affect the autophagy process.

The emergence of brain organoids has revolutionized our understanding of neurodevelopment and neurological diseases by providing an in vitro model system that recapitulates key aspects of human brain development. However, conventional organoid protocols often overlook the role of microglia, the resident immune cells of the central nervous system. Microglia dysfunction is implicated in various neurological disorders, highlighting the need for their inclusion in organoid models. Here, we present a novel method for generating neuroimmune assembloids using human-induced pluripotent stem cell (iPSC)-derived cortical organoids and microglia. Building upon our previous work generating myelinating cortical organoids, we extend our methodology to include the integration of microglia, ensuring their long-term survival and maturation within the organoids. We describe two integration methods: one involving direct addition of microglia progenitors to the organoids and an alternative approach where microglia and dissociated neuronal progenitors are aggregated together in a defined ratio. To facilitate downstream analysis, we also describe a dissociation protocol for single-cell RNA sequencing (scRNA-seq) and provide guidance on fixation, cryosectioning, and immunostaining of assembloid structures. Overall, our protocol provides a comprehensive framework for generating neuroimmune assembloids, offering researchers a valuable tool for studying the interactions between neural cell types and immune cells in the context of neurological diseases.

The ability to alternate between quiescent and proliferating states is a remarkable feature of many types of somatic stem cells. The balance between quiescent and proliferating states is vital for maintenance of stem cells over the lifespan, and its disturbance may lead to premature depletion of the stem cell pool and loss of the tissue regenerative or renewal capacity at later stages of life. The question on how this balance is regulated is of critical importance in stem cell research and biology of aging. Assessment of the balance between quiescent and proliferating states has remained challenged until recently due to the lack of approaches for robust determination of the rate at which stem cells exit reversible cell cycle arrest. Here, we propose a simple method for detection of those stem cells that have entered the division cycle after a prolonged period of quiescence.The method combines cumulative and pulse labeling with thymidine analogues 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). In the discussed labeling scheme, cells that have incorporated only the second label, EdU, are de novo dividing cells. The suggested double labeling method provides quantitative assessment of the rate at which stem cells exit the quiescent state and allows the fates of de novo dividing stem cells to be traced.

The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Isolating pancreatic ductal cells from rats is a critical procedure in pancreatic research, offering valuable insights into pancreatic function, pathology, and potential treatments. The process involves several key steps, beginning with the proper removal of the rat's pancreas, followed by the initiation of the ductal cell isolation procedure. This aims to obtain pure and viable ductal cell populations for further experimentation and analysis.

This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.