Jove-Journal of Visualized Experiments最新文献

Systematic screening of gain- or loss-of-function genetic perturbations can be used to characterize the genetic dependencies and mechanisms of regulation for essentially any cellular process of interest. These experiments typically involve profiling from a pool of single gene perturbations and how each genetic perturbation affects the relative cell fitness. When applied in the context of drug efficacy studies, often called chemo-genetic profiling, these methods should be effective at identifying drug mechanisms of action. Unfortunately, fitness-based chemo-genetic profiling studies are ineffective at identifying all components of a drug response. For instance, these studies generally fail to identify which genes regulate drug-induced cell death. Several issues contribute to obscuring death regulation in fitness-based screens, including the confounding effects of proliferation rate variation, variation in the drug-induced coordination between growth and death, and, in some cases, the inability to separate DNA from live and dead cells. MEDUSA is an analytical method for identifying death-regulatory genes in conventional chemo-genetic profiling data. It works by using computational simulations to estimate the growth and death rates that created an observed fitness profile rather than scoring fitness itself. Effective use of the method depends on optimal tittering of experimental conditions, including the drug dose, starting population size, and length of the assay. This manuscript will describe how to set up a chemo-genetic profiling study for MEDUSA-based analysis, and we will demonstrate how to use the method to quantify death rates in chemo-genetic profiling data.

Cerebrovascular disease is a prevalent condition among the elderly, with its incidence steadily rising. The basilar artery is a critical cerebral vessel that supplies the pons, cerebellum, posterior brain regions, and inner ear. Potassium (K⁺) channel activity plays a significant role in determining vascular tone by regulating the cell membrane potential. Activation of inward rectifying K⁺ (Kir) channels, like other K⁺ channels, leads to cell membrane hyperpolarization and vasodilation. In this study, freshly isolated smooth muscle cells from the basilar artery were used to record Kir currents via the whole-cell patch clamp technique. The effects of 100 µmol/L BaCl2, a Kir channel inhibitor, and 10 µmol/L sodium nitroprusside (SNP), a nitro vasodilator, on Kir channel currents were investigated. The results demonstrated that BaCl2 inhibited Kir channel currents in basilar artery smooth muscle cells, whereas SNP enhanced these currents. This protocol provides a comprehensive guide for preparing freshly isolated arterial smooth muscle cells and recording Kir channel currents using the patch clamp technique, offering a valuable resource for researchers seeking to master this method.

Neuraxial anesthesia is one of the few remaining forms of regional anesthesia that relies on palpation and tactile feedback techniques to facilitate catheterization into the epidural space. Over two decades ago, spine ultrasonography was demonstrated to provide reliable guidance for locating the epidural space. Compared to the palpation technique, preprocedural ultrasonography has been shown to result in fewer needle punctures and fewer traumatic procedures, particularly in patients with abnormal or distorted spine anatomy (e.g., scoliosis, obesity). Despite its utility, the ultrasound-guided neuraxial technique is still marginally used, even for patients with abnormal anatomy. Some experts attribute this to cost, a relatively high success rate without ultrasound, and a lack of technical expertise, which is often tied to formal education and regular practice. Several proponents of the ultrasound technique emphasize that proficiency requires practice on patients with normal spine anatomy, though this training may not be as challenging as once thought. This protocol was designed to help all providers learn the basics of lumbar spine anatomy and how to apply this knowledge clinically. Through a series of videos, we will provide step-by-step instructions for performing neuraxial ultrasonography and offer practical tips for troubleshooting in cases of difficult anatomy.

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young's modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young's modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young's moduli, and generation of Young's modulus maps. Finally, it shows how to determine and isolate Young's modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This method describes the determination of deuterium enrichment of retinol in serum and the estimation of vitamin A stores in the body. The process involves extracting retinol from 0.4 mL of serum using 0.5 mL of 0.85% saline solution, 100 µL of internal standard solution, and 5 mL of chloroform-methanol (2:1 v/v) solution. After centrifugation and removal of the lower chloroform layer, the mixture is dried under nitrogen and resuspended in 0.1 mL of ethanol, and the retinol fraction is separated from other constituents using an HPLC system equipped with a PE C18 column. The retinol fraction can be collected manually or with a fraction collector. Subsequently, the retinol fraction is dried under nitrogen and derivatized with O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 10% trimethylchlorosilane. Finally, labeled and non-labeled retinol isotopes are quantified using a GC-MS system equipped with a 19091z-431 HP-1 methyl siloxane capillary column, employing electron capture negative chemical ionization with helium as the carrier gas and methane as the ionization agent. The ratio of labeled to non-labeled retinol is then used in the Olson, Green, or mass balance equations to estimate vitamin A stores.

Minimally invasive isolated limb perfusion (MI-ILP) is a treatment option for patients with locally advanced melanomas and sarcomas of the extremities. Briefly, the procedure starts with percutaneous access of the femoral or brachial vessels in the diseased extremity. It is then isolated from the rest of the body with a tourniquet. The catheters are connected to a heart-lung machine, and the extremity is perfused with a high dose of melphalan. In the literature, the reported overall and complete response rates for ILP are approximately 80% and 60%, respectively. Our previously reported results using MI-ILP, showed similar response rates. The objective of this manuscript is to provide a step-by-step guide on how to perform an MI-ILP. The purpose of this protocol is to enable local perfusion of the extremities with a high dose of chemotherapy without systemic leakage in a minimally invasive manner.

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0), cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.

Caenorhabditis elegans is a model organism widely used for studying biological processes. Its transparency and small size make it ideal for imaging tissues, cells, and subcellular structures. Traditional flat agar pads for imaging C. elegans limit control over the animal's orientation, restricting views primarily to lateral perspectives. This limitation complicates the visualization of dorsal-ventral structures and reduces image clarity, especially in older animals with increased pigmentation and larger diameters. To overcome these challenges, we developed channeled agarose pads that allow precise control of animal orientation. These channels enable researchers to rotate and fix C. elegans in specified positions, facilitating the simultaneous imaging of multiple structures and improving image resolution by bringing target cells closer to the microscope objective. This is particularly useful for imaging regenerated neuronal fibers after surgery, which may grow in directions difficult to capture with traditional flat agar pads. This method is accessible, as fabricating channeled agar pads requires the same time and materials as flat pads, making it a practical option for most laboratories.

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

Diaphragm dysfunction is a widely recognized concern across numerous medical specialties and clinical settings. Timely and accurate assessment of the diaphragm is vital not only in critically ill patients, where it has a role in weaning from mechanical ventilation and respiratory outcomes, but also in the perioperative arena as a diagnostic tool to detect phrenic nerve function. Diaphragmatic assessment has traditionally utilized fluoroscopy and nerve studies that are time-consuming, costly, and non-portable. Point-of-care ultrasound (POCUS) overcomes these barriers and can be used as a tool for non-invasive screening of diaphragm function. However, POCUS for diaphragmatic dysfunction currently suffers from several issues such as a lack of consensus guidelines, a multiplicity of protocols, and poor interoperator reliability among existing protocols, most notably with the assessment of dome of diaphragm excursion and diaphragmatic thickening. To address these issues, this manuscript reviews the available literature on diaphragmatic POCUS and identifies an image acquisition technique that is both simple to perform and has high interoperator reliability. This technique first describes a qualitative evaluation of diaphragm excursion, followed by a quantitative assessment of the excursion of the zone of apposition. The technique is described stepwise along with all the following: patient positioning, transducer selection, probe placement, image optimization, and interpretation.