Methods in molecular biology最新文献

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

The extracellular matrix (ECM) is a noncellular component of tissues that provides structural and biochemical support to cells. The purpose of decellularization is to provide a tissue-specific niche to preserve the architecture, composition, and signaling molecules of the ECM. The current protocol discusses the standardization of chondrocyte isolation and the preparation of acellular ECM as a bioink additive from human native articular cartilage. Isolated chondrocytes with bioink additives provide a tissue-specific microenvironment. Herein, we discuss a standardized protocol with multiple applications in the area of organ-on-a-chip model development, spheroid formation, microfluidics platform, bioprinting, and tissue engineering. Cartilage tissue engineering is complex owing to the heterogeneous complex proteins, which are a challenge to synthesize; hence, this protocol in many ways offers cues to exploit the acellular ECM for multiple ongoing research studies.

Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event between multiple cell types, and crosstalk between these cell types is an essential part of HSC regulation. Among the cell groups of the niche involved in this process are a group of bone-resident macrophages known as osteomacs (OM). Previously, it was demonstrated that OM and osteoblasts contained within neonatal calvarial cells are critical to maintain hematopoietic function. Additionally, interactions between neonatal calvarial cells and megakaryocytes further enhance this hematopoietic activity. In this chapter, we explore one such interaction involving OM and osteoblasts in the hematopoietic niche. We describe a protocol to isolate OM from both neonatal and adult mice, and subsequently use colony-forming assays to demonstrate their interaction with osteoblasts in maintaining HSC function.

Germ stem cell (GSC) niches are fundamental for the maintenance of the immortal germ cell lineage across generations. In the nematode Caenorhabditis elegans, the simple GSC system has served as an important model for understanding stem cell biology and underlying genetic architecture. GSC niche activity in C. elegans is highly sensitive to subtle environmental and genetic variation. Quantifying variation in the C. elegans GSC niche is therefore essential; however, most methods to do so remain labor-intensive and time-consuming when screening large numbers of individuals. Here, we present a simple and efficient method to estimate the size of the C. elegans GSC niche progenitor pool. This method is ideal for detecting differences in progenitor pool size among different genotypes and environmental treatments during medium- to high-throughput applications such as forward genetic screens and quantitative genetics.

The co-culture method is a simple type of cell culture method used to evaluate the effects of communication between various types of cells in an in vitro setting. In the co-culture method, two or more eukaryotic cell types, or eukaryotic and prokaryotic cells, are cultured together. The co-culture method reflects in vivo cell behaviors and thereby emerges as a pivotal technique with diverse applications in cancer research and cell biology. Two categories of co-culture methods (indirect methods and direct methods) are well known. Direct co-culture methods allow physical contact between the various cell types (juxtacrine signaling). In indirect methods, cells are physically separated into two different populations (for example, using a Transwell) that allow communication only via secretory factors (paracrine signaling). Herein, we focus on the principles of the indirect co-culture method. Nowadays, this method is used to explore the effects of mesenchymal stem cell (MSC) secretome on cancer cells. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell proliferation, invasion, apoptosis, and polarity.

Vernal keratoconjunctivitis (VKC) is a serious eye allergy characterized by poorly understood pathogenic mechanisms and a lack of effective treatments. Autophagy, a process involved in both triggering and suppressing immune and inflammatory responses, plays a role in VKC's pathophysiology. Understanding autophagy's involvement in VKC could lead to new treatment possibilities, such as utilizing specific topical substances to induce or inhibit autophagy and prevent severe complications of this eye condition. In our current protocol, we present a robust methodology established in our laboratory for studying autophagy in primary conjunctival fibroblasts. We assess autophagy through techniques like immunocytochemistry, immunoblotting, and qPCR.