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Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.Laparoscopic transabdominal preperitoneal hernia repair (TAPP) is one of the most widely used methods in inguinal hernia surgery. After the mesh is placed, the peritoneum must be resutured to avoid contact with the tissues and organs in the abdominal cavity. If the peritoneal suture time is too long, the operation and anesthesia time will be prolonged, increasing the burden on the patient. BTK inhibition Moreover, improper suture methods cause serious consequences, such as intestinal obstruction and mesh infection. The straight-needle suture method transforms the three-dimensional spatial configuration of the needle holder and the arc needle tip into a two-dimensional planar structure, which greatly reduces the difficulty of suturing. The three-tailed knot can be anchored at the beginning of the suture by its friction and button effect, which has an exact fixation effect. Thus, the suture does not easily slip, and the time to complete the suturing is shortened. Compared with the traditional suture method, the operator can suture the peritoneum more quickly, beginners can pass through the difficult learning curve faster, and skilled operators can also shorten the total operation time of TAPP to a certain extent. Thus, this suture method is extremely amenable to clinical application.Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging. This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-β, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-β in a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-β-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity >92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.
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