Methods in cell biology最新文献

Among myeloid regulatory cells (MRCs), some particular subsets termed myeloid-derived suppressor cells (MDSCs) have been described. They are suppressor myeloid cells characterized by their ability to regulate innate and adaptive immune responses and known to accumulate in the context of chronic diseases and cancer. The lack of specific markers makes their classification difficult and requires functional studies to distinguish them from other myeloid cells. In this sense, the in vitro analysis of the proliferation of T lymphocytes cultured with MDSCs provides information about the regulatory function of these cells. Here, we provide a detailed protocol to assess the ability of human Mo-MDSCs to suppress T cell proliferation in vitro after obtaining Mo-MDSCs and CD4⁺T cell from peripheral blood.

The functional importance of nitric oxide (NO) in the fields of immunology concerning its antimicrobial, anti-tumoral, anti-inflammatory, and immunosuppressive effects have made it inevitable to study its secretion from various cells. Nitrogen oxide synthase (NOS) is the enzyme responsible for synthesizing NO and its three isoforms function in a cell-dependent manner. NO is oxidized rapidly to Reactive nitrogen oxide species (RNOS) through which the roles of NO are being carried out. One of the major immune cells secreting NO is myeloid-derived suppressor cells (MDSCs). The function of these MDSCs in the suppression of T-cell proliferation as well as T-cell differentiation is found to be dependent on NO secretion. Apart from T-cell suppressive activity, NO is also known to interfere with natural killer (NK) cell functions. A convenient method to estimate NO secretion is by using Griess reagent named after Johann Peter Griess. In this method, NO reacts with the reagents to form a colored azo dye detectable using a microplate reader at a wavelength of 548nm. In this chapter, we summarized the detailed method of estimating NO from MDSCs by the Griess method.

Peritoneal carcinomatosis (PCa) represents a metastatic stage of a disease with unmet therapeutic options. Malignant cells from primary tumors (gastrointestinal or gynecologic malignancies) invade the peritoneal cavity and eventually seed onto peritoneal surfaces, with the omentum being the most common nest area. With a median survival of less than 6 months, PCa has a dismal prognosis that can be improved with treatments only available to a select few individuals with low tumor burden. Thus, the discovery of novel and effective therapies for this disease depends on reliable animal models. Here, we describe a method to generate syngeneic PCa mouse models based on intraperitoneal (i.p.) administration of tumor cells. This model allows to follow-up cancer progression in PCa models from ovarian and colorectal origins monitoring mice bodyweight changes, ascites development and overall survival. Moreover, luciferase-expressing tumor cells can also be used to assess tumor growth after i.p. injection through in vivo bioluminescence quantification. The establishment of reliable, easy-to-monitor and reproducible intraperitoneal syngeneic tumors models, as described here, is the first step to develop cutting-edge therapies against PCa.

The mucosal surface of gastrointestinal tract is lined with epithelial cells that establish an effective barrier between the lumen and internal environment through intercellular junctions, preventing the passage of potentially harmful substances. The "intestinal barrier function" consist of a defensive system that prevent the passage of antigens, toxins, and microbial products, while maintains the correct development of the epithelial barrier, the immune system and the acquisition of tolerance toward dietary antigens and intestinal microbiota. Intestinal morphology changes subsequent to nutritional variations, stress, aging or diseases, which can also affect the composition of the microbiota, altering the homeostasis of the intestine. A growing body of evidence suggests that alterations in intestinal barrier function favor the development of exaggerated immune responses, leading to metabolic endotoxemia, which seems to be the origin of many chronic metabolic diseases such as type 2 diabetes mellitus (T2DM). Although the mechanisms are still unknown, the interaction between dietary patterns, gut microbiota, intestinal mucosa, and metabolic inflammation seems to be a key factor for the development of T2DM, among other diseases. This chapter details the different techniques that allow evaluating the morphological and molecular alterations that lead of the intestinal barrier dysfunction in a T2DM experimental model. To induce both diabetic metabolic disturbances and gut barrier disruption, Wistar rats were fed a high-saturated fat and high-cholesterol diet and received a single dose of streptozotocin/nicotinamide. This animal model may contribute to clarify the understanding of the role of intestinal barrier dysfunction on the late-stage T2DM etiology.

Aneuploidy is a condition in which cells have an abnormal number of chromosomes that is not a multiple of the haploid complement. It is known that aneuploidy has detrimental consequences on cell physiology, such as genome instability, metabolic and proteotoxic stress and decreased cellular fitness. Importantly, aneuploidy is a hallmark of tumors and it is associated with resistance to chemotherapeutic agents and poor clinical outcome. To shed light into how aneuploidy contributes to chemoresistance, we induced chromosome mis-segregation in human cancer cell lines, then treated them with several chemotherapeutic agents and evaluated the emergence of chemoresistance. By doing so, we found that elevation of chromosome mis-segregation promotes resistance to chemotherapeutic agents through the expansion of aneuploid karyotypes and subsequent selection of specific aneuploidies essential for cellular viability under those stressful conditions. Here, we describe a method to generate aneuploid cell populations and to evaluate their resistance to anti-cancer agents. This protocol has been already successfully employed and can be further utilized to accelerate the exploration of the role of aneuploidy in chemoresistance.

Multiple DNA repair pathways and biological responses to DNA damage have evolved to protect cells from various types of lesions to which they are subjected. Although DNA repair systems are mechanistically distinct, all process the damaged region and then insert new bases to fill the gap. In 1969, Robert Painter developed an assay called "unscheduled" DNA synthesis (UDS), which measures DNA repair synthesis as the uptake of radiolabeled DNA precursors distinct from replicative synthesis. Contemporary detection of nascent DNA during repair by next-generation sequencing grants genome-wide information about the nature of lesions that threaten genome integrity. Recently, we developed the SAR-seq (synthesis associated with repair sequencing) method, which provides a high-resolution view of UDS. SAR-seq has been utilized to map programmed DNA repair sites in non-dividing neurons, replication initiation zones, monitor 53BP1 function in countering end-resection, and to identify regions of the genome that fail to complete replication during S phase but utilize repair synthesis during mitosis (MiDAS). As an example of SAR-seq, we present data showing that sites replicated during mitosis correspond to common fragile sites, which have been linked to tumor progression, cellular senescence, and aging.

Replication stress risks genomic integrity. Depending on the level, replication stress can lead to slower progression through S phase and entry into G2 phase with DNA damage. In G2 phase, cells either recover and eventually enter mitosis or permanently withdraw from the cell cycle. Here we describe a method to detect cell cycle distribution, replication stress and cell cycle exit from G2 phase using fluorescence microscopy. We provide a script to automate the analysis using ImageJ. The focus has been to make a script and setup that is accessible to people without extensive computer knowledge.

Caenorhabditis elegans is a nematode that has been used as an animal model for almost 50years. It has primitive and simple tissues and organs, making it an ideal model for studying neurological pathways involved in neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD). C. elegans has conserved neurological pathways and is able to mimic human diseases, providing valuable insights into the human disease phenotype. This methodological review presents current approaches to generate neurodegenerative-like models of AD and PD in C. elegans, and evaluates the experiments commonly used to validate the diseases. These experimental approaches include assessing survival, fertility, mobility, electropharyngeogram assays, confocal mitochondrial imaging, RNA extraction for qRT-PCR or RT-PCR, and rate of defecation. This review also summarizes the current knowledge acquired on AD and PD using the aforementioned experimental approaches. Additionally, gaps in knowledge and future directions for research are also discussed in the review.

Repeat and structure-prone DNA sequences comprise a large proportion of the human genome. The instability of these sequences has been implicated in a range of diseases, including cancers and neurodegenerative disorders. However, the mechanism of pathogenicity is poorly understood. As such, further studies on repetitive DNA are required. Cloning and maintaining repeat-containing substrates is challenging due to their inherent ability to form non-B DNA secondary structures which are refractory to DNA polymerases and prone to undergo rearrangements. Here, we describe an approach to clone and expand tandem-repeat DNA without interruptions, thereby allowing for its manipulation and subsequent investigation.