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Past Issue in 2023
Volume: 13, Issue: 9
Biochemistry
A Simple, Reproducible Procedure for Chemiluminescent Western Blot Quantification
Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture. However, there is no clear and common procedure to quantify the results obtained, resulting in variations due to the different software and protocols used in each laboratory. Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. Images were processed with ImageJ and subsequently compared using R software. The result is a linear regression model in which we use the slope of the signal increase within the combined linear range of detection to compare between samples. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way. Graphical overview
Biological Engineering
Protocol for 3D Bioprinting Mesenchymal Stem Cell–derived Neural Tissues Using a Fibrin-based Bioink
Three-dimensional bioprinting utilizes additive manufacturing processes that combine cells and a bioink to create living tissue models that mimic tissues found in vivo. Stem cells can regenerate and differentiate into specialized cell types, making them valuable for research concerning degenerative diseases and their potential treatments. 3D bioprinting stem cell–derived tissues have an advantage over other cell types because they can be expanded in large quantities and then differentiated to multiple cell types. Using patient-derived stem cells also enables a personalized medicine approach to the study of disease progression. In particular, mesenchymal stem cells (MSC) are an attractive cell type for bioprinting because they are easier to obtain from patients in comparison to pluripotent stem cells, and their robust characteristics make them desirable for bioprinting. Currently, both MSC bioprinting protocols and cell culturing protocols exist separately, but there is a lack of literature that combines the culturing of the cells with the bioprinting process. This protocol aims to bridge that gap by describing the bioprinting process in detail, starting with how to culture cells pre-printing, to 3D bioprinting the cells, and finally to the culturing process post-printing. Here, we outline the process of culturing MSCs to produce cells for 3D bioprinting. We also describe the process of preparing Axolotl Biosciences TissuePrint - High Viscosity (HV) and Low Viscosity (LV) bioink, the incorporation of MSCs to the bioink, setting up the BIO X and the Aspect RX1 bioprinters, and necessary computer-aided design (CAD) files. We also detail the differentiation of 2D and 3D cell cultures of MSC to dopaminergic neurons, including media preparation. We have also included the protocols for viability, immunocytochemistry, electrophysiology, and performing a dopamine enzyme-linked immunosorbent assay (ELISA), along with the statistical analysis.Graphical overview
Cell Biology
Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse
Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.
Developmental Biology
E15.5 Mouse Embryo Micro-CT Using a Bruker Skyscan 1172 Micro-CT
X-ray computed microtomography (µCT) is a powerful tool to reveal the 3D structure of tissues and organs. Compared with the traditional sectioning, staining, and microscopy image acquisition, it allows a better understanding of the morphology and a precise morphometric analysis. Here, we describe a method for 3D visualization and morphometric analysis by µCT scanning of the embryonic heart of iodine-stained E15.5 mouse embryos.
Drug Discovery
Implementation of a Drug Screening Platform to Target Gch1 Expression in Injured Mouse Dorsal Root Ganglion Neurons
Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene, Gch1, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers. Gch1 is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced Gch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell–derived sensory neurons.Graphical overview
Medicine
A Novel Non-invasive Qualitative Assay Using Urinary Fluorescence Imaging to Assess Kidney Disease
In patients with chronic kidney disease, it is necessary to identify the etiology of glomerular disease. Renal biopsy is the gold standard for assessing the underlying pathology; however, it has the risk of potential complications. We have established a urinary fluorescence imaging technique to assess enzymatic activity using an activatable fluorescent probe targeting two enzymes: gamma-glutamyl transpeptidase and dipeptidyl-peptidase. The urinary fluorescence images can be easily obtained by adding an optical filter to the microscope with short incubation of the fluorescent probes. Urinary fluorescence imaging could help to assess underlying etiologies of kidney diseases and is a potential non-invasive qualitative assessment technique for kidney diseases in patients with diabetes.Key features• Non-invasive assessment of kidney disease.• Urinary fluorescent imaging with enzyme-activatable fluorescent probes.• Enables differentiation of diabetic kidney disease and glomerulonephritis.
Microbiology
Novel Antibody-independent Method to Measure Complement Deposition on Bacteria
During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such as Staphylococcus aureus to overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such as S. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and both Lactococcus lactis and S. aureus.
Neuroscience
Long-term in toto Imaging of Cellular Behavior during Nerve Injury and Regeneration
Accidental wounding of the peripheral nervous system leads to acute neural dysfunction. Normally, chronic deficits are overcome because peripheral nerves naturally regenerate. However, various genetic and metabolic defects can impair their natural regenerative capacity, which may be due to neuron-extrinsic mechanisms. Therefore, characterizing the behavior of multiple cells during nerve injury and repair in vivo is a pressing need in regenerative medicine. Here, we detail a method for precise wounding of sensory axons in zebrafish, followed by high-resolution in toto long-term quantitative videomicroscopy of neurons, Schwann cells, and macrophages. This protocol can be easily adapted to study the effects of targeted genetic or metabolic disruptions in zebrafish and other suitable organisms, as well as for screening pharmacological agents with therapeutic potential.Graphical overview
Simultaneous Microendoscopic Calcium Imaging and EEG Recording of Mouse Brain during Sleep
Sleep is a conserved biological process in the animal kingdom. Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology, important for the development of new treatments for insomnia and other sleep-related disorders. Yet, brain circuits controlling this process remain poorly understood. A key technique in sleep research is to monitor in vivo neuronal activity in sleep-related brain regions across different sleep states. These sleep-related regions are usually located deeply in the brain. Here, we describe technical details and protocols for in vivo calcium imaging in the brainstem of sleeping mice. In this system, sleep-related neuronal activity in the ventrolateral medulla (VLM) is measured using simultaneous microendoscopic calcium imaging and electroencephalogram (EEG) recording. By aligning calcium and EEG signals, we demonstrate that VLM glutamatergic neurons display increased activity during the transition from wakefulness to non-rapid eye movement (NREM) sleep. The protocol described here can be applied to study neuronal activity in other deep brain regions involved in REM or NREM sleep.
Assessment of Chemosensory Response to Volatile Compounds in Healthy, Aged, and Neurodegenerative Caenorhabditis elegans Models
A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy individuals and Caenorhabditis elegans models associated with neurodegenerative diseases.
Plant Science
Modified Pseudo-Schiff Propidium Iodide for Staining the Shoot Apical Meristem in Arabidopsis
Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.