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Past Issue in 2021
Volume: 11, Issue: 6
Biochemistry
Monitoring Real-time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism
RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes. Observing RNA structural changes in real-time over a range of temperatures is therefore highly desirable. A variety of approaches exists that probe RNA secondary structures, but many of these either require large amount and/or extensive processing of the RNA or cannot be applied under physiological conditions, rendering the observation of structural dynamics over a range of temperatures difficult. Here, we describe the use of a dually fluorescently labelled RNA oligonucleotide (containing the predicted hairpin structure) that can be used to monitor subtle RNA-structural dynamics by Förster Resonance Energy Transfer (FRET) at different temperatures with RNA concentration as low as 200 nM. FRET efficiency varies as a function of the fluorophores’ distance; high efficiency can thus be correlated to a stable hairpin structure, whilst a reduction in FRET efficiency reflects a partial opening of the hairpin or a destabilisation of this structure. The same RNA sequence can also be used for Circular Dichroism spectroscopy to observe global changes of RNA secondary structure at a given temperature. The combination of these approaches allowed us to monitor RNA structural dynamics over a range of temperatures in real-time and correlate structural changes to plant biology phenotypes.Graphic abstract:Monitoring temperature-dependent RNA structural dynamics using Förster Resonance Energy Transfer (FRET)
Biological Engineering
Multilayered Fabrication Assembly Technique to Engineer a Corneal Stromal Equivalent
Tissue engineering has emerged as a strategy to combat the donor shortage of human corneas for transplantation. Synthetic corneal substitutes are currently unable to support the normal phenotype of human cells and so decellularized animal corneas have been deployed to more closely provide the topographical and biochemical cues to promote cell attachment and function. Although full thickness decellularized corneas can support corneal cells, the cells are slow to populate the scaffold and density declines from the surface. To avoid these problems, this protocol describes the stacking of alternate layers of decellularized porcine corneal sheets and cell-laden collagen hydrogel to produce a corneal construct. The sheets are obtained by cryosectioning porcine corneas, decellularizing them with detergents and nucleases and finally air drying for storage and ease of manufacture. Corneal stromal cells are then encapsulated in a collagen type I solution and cast between these sheets. This protocol presents a rapid method to ensure high cellularity throughout the construct using tissue-derived materials alone.Graphic abstract:Overview of main process to obtain corneal stromal equivalents
Cancer Biology
Preparation of an Orthotopic, Syngeneic Model of Lung Adenocarcinoma and the Testing of the Antitumor Efficacy of Poly(2-oxazoline) Formulation of Chemo-and Immunotherapeutic Agents
Tumor xenograft models developed by transplanting human tissues or cells into immune-deficient mice are widely used to study human cancer response to drug candidates. However, immune-deficient mice are unfit for investigating the effect of immunotherapeutic agents on the host immune response to cancer (Morgan, 2012). Here, we describe the preparation of an orthotopic, syngeneic model of lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), to study the antitumor effect of chemo and immunotherapeutic agents in an immune-competent animal. The tumor model is developed by implanting 344SQ LUAD cells derived from the metastases of KrasG12D; p53R172HΔG genetically engineered mouse model into the left lung of a syngeneic host (Sv/129). The 344SQ LUAD model offers several advantages over other models: 1) The immune-competent host allows for the assessment of the biologic effects of immune-modulating agents; 2) The pathophysiological features of the human disease are preserved due to the orthotopic approach; 3) Predisposition of the tumor to metastasize facilitates the study of therapeutic effects on primary tumor as well as the metastases (Chen et al., 2014). Furthermore, we also describe a treatment strategy based on Poly(2-oxazoline) micelles that has been shown to be effective in this difficult-to-treat tumor model (Vinod et al., 2020b).
Intestinal Co-culture System to Study TGR5 Agonism and Gut Restriction
The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to assess both the pharmacodynamics of TGR5 agonists and the toxicity of compounds to the intestinal monolayer. As a proof of concept, we demonstrate the use of the protocol in evaluating properties of naturally occurring bile acid metabolites that are potent TGR5 agonists. This protocol is adapted from Chaudhari et al. (2021).
Preparation and Characterization of Poly(2-oxazoline) Micelles for the Solubilization and Delivery of Water Insoluble Drugs
Many new drug development candidates are highly lipophilic compounds with low water solubility. This constitutes a formidable challenge for the use of such compounds for cancer therapy, where high doses and intravenous injections are needed (Di et al., 2012). Here, we present a poly(2-oxazoline) polymer (POx)-based nanoformulation strategy to solubilize and deliver hydrophobic drugs. POx micelles are prepared by a simple thin-film hydration method. In this method, the drug and polymer are dissolved in a common solvent and allowed to mix, following which the solvent is evaporated using mild heating conditions to form a thin film. The micelles form spontaneously upon hydration with saline. POx nanoformulation of hydrophobic drugs is unique in that it has a high drug loading capacity, which is superior to micelles of conventional surfactants. Moreover, multiple active pharmaceutical ingredients (APIs) can be included within the same POx micelle, thereby enabling the codelivery of binary as well as ternary drug combinations (Han et al., 2012; He et al., 2016).
Cell Biology
Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells
More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway in order to mature and be properly sorted to their final destinations. Among several methodologies that describe live-cell monitoring of vesicles, the Retention Using Selective Hooks (RUSH) system is a powerful one that allows to visualize cargo trafficking under physiological conditions. The present protocol describes a method to use the RUSH system in live-cell microscopy and a subsequent quantitative analysis of cargo vesicles to dissect protein trafficking. In brief, HeLa cells are transiently transfected with an MMP2-RUSH construct and vesicle trafficking is evaluated by wide-field microscopy, recording videos in 1-min time frames for 45 min. We also present a quantitative approach that can be used to identify kinetics of uncharacterized protein cargo, as well as to evaluate with more detail processes such as ER-to-Golgi vesicle trafficking.Graphic abstract:Live-cell RUSH: a tool to monitor real-time protein trafficking in the secretory pathway
Immunology
Murine Monocyte and Macrophage Culture
Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).Graphic abstract:Figure 1. Experimental outline depicting steps for murine monocyte and macrophage culture
Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages.Graphical abstract:Experimental workflow for in vivo macrophage-specific depletion by liposomal clodronate in larval zebrafish
An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration
In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.
Quantitative Measurement of Mucolytic Enzymes in Fecal Samples
The mucus layer in the gastrointestinal tract covers the apical surface of intestinal epithelial cells, protecting the mucosal tissue from enteric pathogen and commensal microorganisms. The mucus is primarily composed of glycosylated protein called mucins, which are produced by goblet cells, a type of columnar epithelial cells in the intestinal tract. Defective mucin barrier facilitates infection caused by enteric pathogen and triggers inflammation due to invasion of commensal or opportunistic pathogens into the intestinal epithelial mucosa. Several bacterial species in the gut produce enzymes that are capable of degradation of the mucus. Defective mucin production or increased abundance of mucolytic bacteria are clinically linked to inflammatory bowel disease. Measurement of mucolytic enzymes in the feces, therefore, can be implicated in clinical and experimental research on intestinal disorders. Here, we describe a step-by-step procedure for the measurement of the mucolytic enzyme activity in fecal samples.
Microbiology
A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells
Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.
Detection and Quantification of African Swine Fever Virus in MA-104 Cells
Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus aethiops) kidney epithelial cells, supports the detection of ASFV from field samples with a sensitivity comparable to that of primary swine macrophages. Collection of swine blood or lungs is time costing, which is often not readily available in most veterinary diagnostic laboratories. MA-104 cells could thus be used as substitute for primary swine macrophages to save significant lead time by avoiding the production of primary swine macrophages.
Molecular Biology
Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples
During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi et al., 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.
Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies. Because of this, we developed a real-time base excision repair assay with a specially designed double-stranded reporter oligonucleotides to measure the activity of hOGG1 in lysates of isolated mitochondria. This system presented here differs from the classical assays, in which an endpoint determination is performed via a denaturing acrylamide gel, by the possibility to measure the hOGG1 activity in real-time. In addition, to determine the activity of each enzymatic step (N-glycosylase and AP-lyase activity) of this bifunctional enzyme, a melting curve analysis can also be performed. After isolation of mitochondria from human fibroblasts using various centrifugation steps, they are lysed and then incubated with specially designed reporter oligonucleotides. The subsequent measurement of hOGG1 activity is performed in a conventional real-time PCR system.
Neuroscience
Investigate Synaptic Vesicles Mobility in Neuronal Culture Axons by FRAP Imaging
Synaptic vesicles (SVs) are clustered in the presynaptic terminals and consistently trafficking along axons. Based on their release features, SVs are classified into different “pools”. Imaging of SVs that are traveling among multiple presynaptic terminals has helped define a new pool named “SV super-pool”. Here we describe a Fluorescent Recovery After Photobleaching (FRAP) approach to elucidate the relationship between SVs from the super-pool with SV clusters at presynaptic terminals. This method is powerful to investigate SV mobility regulation mechanisms.
Ligand and Carbohydrate Engagement (LACE) Assay and Fluorescence Quantification on Murine Neural Tissue
The interaction between cell surface heparan sulphate and diffusible ligands such as FGFs is of vital importance for downstream signaling, however, there are few techniques that can be used to investigate this binding event. The ligand and carbohydrate engagement (LACE) assay is a powerful tool which can be used to probe the molecular interaction between heparan sulphate and diffusible ligands and can detect changes in binding that may occur following genetic or pharmacological intervention. In this protocol we describe an FGF17:FGFR1 LACE assay performed on embryonic mouse brain tissue. We also describe the method we have used to quantify changes in fluorescent LACE signal in response to altered HS sulphation.
A Time Duration Discrimination Task for the Study of Elapsed Time Processing in Rats
Space and time are both essential features of episodic memory. However, while spatial tasks have been used effectively to study the behavioral relevance of place cells, the behavioral paradigms utilized for the study of time cells have not used time duration as a variable that animals need to be aware of to solve the task. In order to evaluate how time flow is coded into memory, time duration needs to be a variable that animals use to solve the behavioral task. This protocol describes a novel behavioral paradigm, the time duration discrimination (TDD) task, which is designed to directly investigate the neurological mechanisms that underlie temporal processing. During the TDD task, rats navigate around a Figure-8 Maze, which contains a rectangular track with a central arm and a delay box at the end of the central arm. While confined to the delay box, rats experience a 10- or 20-second time delay, during which a tone will play for the duration of the 10- or 20-second delay. When the delay box opens, the rat will choose whether to turn left or right out of the delay box and receive a reward for the correct choice (e.g., 10 seconds = left turn; 20 seconds = right turn). By directly manipulating elapsed time, we can better explore the behavioral relevance of hippocampal time cells and whether the time-dependent activity seen in physiological recordings of hippocampal neurons reflects a neuronal representation of time flow that can be used by the animal for learning and storing memories.Graphic abstract:Elapsed time duration discrimination in rats