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Past Issue in 2021
Volume: 11, Issue: 8
Biochemistry
Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance.Graphic abstract:Schematic overview of the workflow described in this protocol
Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase
(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely uncharacterized due to the absence of informative activity assays. Previously, a radioactive assay and reducing end modification have been used to characterize GS activity. The radioactive assay determines only the total amount of glucan formed through glucose incorporation and does not report the length of the polymers produced. The glucan length has been characterized by reducing end modification, but this method is unsuitable for mechanistic studies due to the very high detection limit of millimolar amounts and the labor intensiveness of the technique. Consequently, fundamental aspects of GS catalysis, such as the polymer length specificity, remain ambiguous. We have developed a size exclusion chromatography (SEC)-based method that allows detailed functional and mechanistic characterization of GS. The approach harnesses the pH-dependent solubility of (1,3)-β-d-glucan, where (1,3)-β-d-glucan forms water-soluble random coils under basic pH conditions, and can be analyzed by SEC using pulsed amperometric detection (PAD) and radioactivity counting (RC). This approach allows quantitative characterization of the total amount and length of glucan produced by GS with minimal workup and a d-glucose (Glc) detection limit of ~100 pmol. Consequently, this approach was successfully used for the kinetic characterization of GS, providing the first detailed mechanistic insight into GS catalysis. Due to its sensitivity, the assay is applicable to the characterization of GS from any fungi and can be adapted to study other polysaccharide synthases.
Cancer Biology
A Robust Mammary Organoid System to Model Lactation and Involution-like Processes
The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer.Graphic abstract:Mammary gland organoid isolation and culture procedures
Cell Biology
AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells
Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed (Bouquier et al., 2020). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells.Graphic abstract:AIMTOR protocol overview from seeding cells to live BRET recording
Developmental Biology
Development of a Chemical Reproductive Aging Model in Female Rats
Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioral changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, around 45-50 years old, women are more likely to express mood disorders, anxiety, irritability and vasomotor symptoms. The current animal models of reproductive aging do not successfully replicate human perimenopause and the gradual changes that occur in this phase. While the traditional rat model of menopause involves ovariectomy or surgical menopause consisting of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones, natural or transitional menopause is achieved by the selective loss of ovarian follicles (perimenopause period). However, the natural aging rodent (around 18-24 months) model fails to reach very low estrogen concentrations and overlaps the processes of somatic and reproductive aging. The chronic exposure of young rodents to 4-vinylcyclohexene diepoxide (VCD) is a well-established experimental model for perimenopause and menopause studies. VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia (apoptosis). The VCD, ovary-intact or accelerated ovarian failure (AOF) model is the experimental model that most closely matches natural human progression to menopause mimicking both hormonal and behavioral changes typically manifested by women in perimenopause.Graphical abstract:The female reproductive system is regulated by a series of neuroendocrine events controlled by central and peripheral components. (A). The mechanisms involved in this control are extremely complex and have not yet been fully clarified. In female mammals whose ovulation (the most important event in a reproductive cycle) occurs spontaneously, reproductive success is achieved through the precise functional and temporal integration of the hypothalamus-pituitary-ovary (HPO) axis. (B). In women, loss of fertility appears to be primarily associated with exhaustion of ovarian follicles, and this process occurs progressively until complete follicular exhaustion marked by the final menstrual period (FMP). (C). While in female rodents, reproductive aging seems to begin as a neuroendocrine process, in which changes in hypothalamic/pituitary function appear independently of follicular atresia. The traditional rat model of menopause, ovariectomy or surgical menopause consists of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones. (D). The chronic exposure (15-30 days) to the chemical compound 4-vinylcyclehexene diepoxide (VCD) in young rodents accelerates gradual failure of ovarian function by progressive depletion of primordial and primary follicles, but retains residual ovarian tissue before brain alterations that occurs in women in perimenopause. Low doses of VCD cause the selective destruction of the small preantral follicles of the ovary without affecting other peripheral tissues.
Production of Phenotypically Uniform Human Cerebral Organoids from Pluripotent Stem Cells
Recent advances in stem cell technology have allowed researchers to generate 3D cerebral organoids (COs) from human pluripotent stem cells (hPSCs). Indeed, COs have provided an unprecedented opportunity to model the developing human brain in a 3D context, and in turn, are suitable for addressing complex neurological questions by leveraging advancements in genetic engineering, high resolution microscopy, and tissue transcriptomics. However, the use of this model is limited by substantial variations in the overall morphology and cellular composition of organoids derived from the same pluripotent cell line. To address these limitations, we established a robust, high-efficiency protocol for the production of consistent COs by optimizing the initial phase of embryoid body (EB) formation and neural induction. Using this protocol, COs can be reproducibly generated with a uniform size, shape, and cellular composition across multiple batches. Furthermore, organoids that developed over extended periods of time (3–6 months) showed the establishment of relatively mature features, including electrophysiologically active neurons, and the emergence of oligodendrocyte progenitors. Thus, this platform provides a robust experimental model that can be used to study human brain development and associated disorders.Graphic abstract:Overview of cerebral organoid development from pluripotent stem cells
Microbiology
Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal activity; yet, these are not definitive indicators. As a result, the presence of integrase genes is often used as a hallmark for lysogenic behavior; however, the accuracy of genetic screening for lysogeny depends on the quality of the extraction, sequencing and assembly of the phage genome, and database comparison. The present protocol describes a simple phenotypic test that can be used to screen therapeutically relevant phages for lysogenic behavior. This test relies on the identification of spontaneous phage release from their lysogenized host and can be reliably used in cases where no sequencing data are available. The protocol does not require specialized equipment, is not work-intensive, and is broadly applicable to any phage with an easily culturable bacterial host, making it particularly amenable to settings with limited resources.Graphical abstract:Screening pipeline for lysogen activity of a given phage
A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels.Graphic abstract:Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a).
Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.
Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay
The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated β-lactamase cleaves the β-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.
Neuroscience
Single or Repeated Ablation of Mouse Olfactory Epithelium by Methimazole
Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature makes the OE a useful model for studying neurogenesis and neuronal differentiation, with the possibility for systemic as well as local administration of various compounds and infectious agents that may interfere with these cellular processes. Several different chemical compounds have been shown to cause toxic injury to the OE, which can be used for OE ablation. We, and others, have found that the systemic administration of the hyperthyroid drug, methimazole, reliably causes olfactotoxicity as a side effect. Here, we outline an OE lesioning protocol for single or repeated ablation by methimazole. A single methimazole administration can be used to study neuroepithelial regeneration and stem cell activation, while repeated ablation and regeneration of OE enable the study of tissue stem cell exhaustion and generation of tissue metaplasia.
Single-unit Recording in Awake Behaving Non-human Primates
Non-human primates (NHPs) have been widely used as a species model in studies to understand higher brain functions in health and disease. These studies employ specifically designed behavioral tasks in which animal behavior is well-controlled, and record neuronal activity at high spatial and temporal resolutions while animals are performing the tasks. Here, we present a detailed procedure to conduct single-unit recording, which fulfils high spatial and temporal resolutions while macaque monkeys (i.e., widely used NHPs) perform behavioral tasks in a well-controlled manner. This procedure was used in our previous study to investigate the dynamics of neuronal activity during economic decision-making by the monkeys. Monkeys’ behavior was quantitated by eye position tracking and button press/release detection. By inserting a microelectrode into the brain, with a grid system in reference to magnetic resonance imaging, we precisely recorded the brain regions. Our experimental system permits rigorous investigation of the link between neuronal activity and behavior.
Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a light-activated, genetically encoded redox sensor that can be activated with precise spatial and temporal control. Here we describe a protocol for the expression and activation of the CofActor system in dissociated hippocampal neuron cultures prepared from newborn mice. Cultures were transfected with Lipofectamine on the fifth day in vitro (DIV5), then exposed to cellular stress inducing stimuli, leading to the formation of actin-cofilin rods that can be observed using live cell imaging techniques. The protocol described here allows for studies of stress-related cytoskeletal dysregulation in live neurons exposed to neurodegenerative stimuli, such as toxic Aβ42 oligomers. Moreover, expression of the sensor in neurons isolated from transgenic mouse models of AD and/or mice KO for proteins involved in AD can advance our understanding of the molecular basis of early cytoskeletal dysfunctions associated with neurodegeneration.
A Method to Quantify Drosophila Behavioral Activities Induced by GABAA Agonist Muscimol
Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria. As a potent GABAA receptor agonist, muscimol suppresses the activity of the central nervous system, reduces anxiety and induces sleep. We investigated the effects of muscimol on Drosophila behavior. Drosophila behavioral assays are powerful tools that are used to assess neural functions by focusing on specific changes in selected behavior, with the hypothesis that this behavioral change is due to alteration of the underlying neural function of interest. In this study, we developed a comparatively simple and cost-effective method for feeding adult flies muscimol, a pharmacologically active compound, and for quantifying the phenotypes of “resting” and “grooming+walking”. This protocol may provide researchers with a convenient method to characterize small molecule-induced behavioral output in flies.
Systems Biology
Computational Analysis and Phylogenetic Clustering of SARS-CoV-2 Genomes
COVID-19, the disease caused by the novel SARS-CoV-2 coronavirus, originated as an isolated outbreak in the Hubei province of China but soon created a global pandemic and is now a major threat to healthcare systems worldwide. Following the rapid human-to-human transmission of the infection, institutes around the world have made efforts to generate genome sequence data for the virus. With thousands of genome sequences for SARS-CoV-2 now available in the public domain, it is possible to analyze the sequences and gain a deeper understanding of the disease, its origin, and its epidemiology. Phylogenetic analysis is a potentially powerful tool for tracking the transmission pattern of the virus with a view to aiding identification of potential interventions. Toward this goal, we have created a comprehensive protocol for the analysis and phylogenetic clustering of SARS-CoV-2 genomes using Nextstrain, a powerful open-source tool for the real-time interactive visualization of genome sequencing data. Approaches to focus the phylogenetic clustering analysis on a particular region of interest are detailed in this protocol.