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Past Issue in 2021
Volume: 11, Issue: 23
Biological Engineering
A low-cost Portable Device to Deliver Smoke, Volatile or Vaporized Substances to Drosophila melanogaster , Useful for Research and/or Educational Assays
Drosophila melanogaster has been used to test drugs of abuse, substances with potential benefits for medical purposes, as well as contaminants and hazardous volatile compounds. This model has also been used for the characterization of behavioral changes, physiopathological consequences, and subcellular mechanisms of the use of cocaine, methamphetamines, ethanol, nicotine, cannabinoids, toluene, and other airborne volatile organic compounds. When testing these substances, routes of administration are important to define. Admixing the test compounds with water or food is one suitable option in many cases, but the inhalation route is especially suitable when the administration of one or more volatile compounds is desired. One advantage of the administration of substances via the inhalation route is its rapid exchange and distribution throughout the cuticle and the tracheal system. In addition, this route allows treating a large group of individuals simultaneously. Moreover, the inhalation route is frequently used to administer different drugs to humans. A good model system shares physiology and molecular pathways with humans, and D. melanogaster possesses almost 75% homologous genes associated with human diseases. Methodologies to deliver the abovementioned substances usually include customized devices. Herein, we focus on the development of a low-cost customized device useful to deliver smoke or vaporizable compounds to D. melanogaster. This approach might be applied for acute or chronic exposure to vaporized substances. In particular, our device was utilized for testing cigarette smoke and vaporized cannabis extract on cardiac performance of adult individuals during chronic treatment. We are describing how to set up this low-cost portable device, useful for research and/or educational assays, taking advantage of the amenability of D. melanogaster to test different compounds in relatively short periods, and especially including a large number of individuals at the same time.Graphic abstract: Custom-made device useful for inhalation pathway assays in Drosophila melanogaster.
Cell Biology
Quantitative Determination of Primary Cilia Protein Distribution Using Immunofluorescence Staining and MATLAB Analysis
Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function.Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images.
Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing
In vivo erythropoiesis occurs in the erythroblast island niche (EBI), comprising of a central macrophage that attaches to and aids the maturation of erythroid progenitors into mature reticulocytes. Macrophages in hematopoietic tissue such as embryonic fetal liver are heterogeneous and express the cell surface protein F4/80. Earlier methods of isolating F4/80+ macrophages from hematopoietic tissue relied on FACS sorting, but the relatively low numbers of F4/80+ cells obtained after FACS sometimes led to poor RNA quality. Additionally, since EBI macrophages are attached to erythroblasts, care must be taken to avoid contamination with bound erythroblasts. We have developed a novel method for isolating F4/80+ cells from E13.5 mouse fetal liver using magnetic nanoparticles, which can be performed on the lab bench. During cell suspension and homogenization, we also add a peptide that disrupts erythroid macrophage interactions and generates F4/80+ single cells free of erythroid contamination. Thus, our protocol generates a population enriched in F4/80+ cells that are healthy and ready for sensitive techniques such as single cell sequencing.
In vitro Fluid Shear Stress Induced Sclerostin Degradation and CaMKII Activation in Osteocytes
Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/β-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.
A Simple Method for in situ Quantification of Cells on Carriers
The technology of cell carriers was developed as a response to the need for high cell density to enable higher production levels in cell-based production processes. To follow the production process, quantifying the number of cells on these carriers is required, as well as tracking their viability and proliferation. However, owing to various carriers’ unique structures, tracking the cells is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, which is tedious and counts both live and dead cells. A few other techniques have been developed, but they are all specific to a carrier type and involve specialized equipment. Here, we describe a broad ranging method for counting cells on carriers. The method is based on the Alamar blue dye, a well-known, common marker for cell activity. No separation of the cells from the carriers is needed, nor is any specialized equipment. The method is simple and rapid, and provides comprehensive details necessary for control of production processes in cells. This method can be easily implemented in any cell-based process and other unique platforms for measuring growth of cells.Graphic abstract: Schematic of the in situ quantification method.
Developmental Biology
In vivo Imaging of Calcium Activities from Pancreatic β-cells in Zebrafish Embryos Using Spinning-disc Confocal and Two-photon Light-sheet Microscopy
Visualizing the function of pancreatic β-cells in vivo has been a long-sought goal for β-cell researchers. Unlike imaging of β-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of β-cells in their native physiological microenvironment in vivo. Here, we detail a protocol for real-time visualization of individual β-cell function in vivo in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg (ins:Rcamp1.07) with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous β-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote β-cell function or maturation using a living whole organism system.
Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc
Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple ex-vivo assay for identifying potential microtubule regulators in the wing imaginal disc of Drosophila melanogaster. This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, we designed an assay using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, we can test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in Drosophila. This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms.Graphic abstract: Graphical summary of Ex-vivo microtubule stability assay using Drosophila wing disc.
Immunology
Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses
Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection (Jakic et al., 2021). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection.Graphic abstract: Figure 1. Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing Listeria monocytogenes) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.
Microbiology
A Phenotypic Screen for the Liver Stages of Plasmodium vivax
Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite’s liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype.Graphical abstract: Overview of P. vivax liver stage screening assay performed at the Institute Pasteur of Cambodia.
A Retro-orbital Sinus Injection Mouse Model to Study Early Events and Reorganization of the Astrocytic Network during Pneumococcal Meningitis
Pneumococcal (PN) meningitis is a life-threatening disease with high mortality rates that leads to permanent neurological sequelae. Studies of the process of bacterial crossing of the blood brain barrier (BBB) are hampered by the lack of relevant in vitro and in vivo models of meningitis that recapitulate the human disease. PN meningitis involves bacterial access to the bloodstream preceding translocation across the BBB. A large number of PN meningitis models have been developed in mice, with intravenous administration via the lateral tail vein representing the main way to study BBB crossing by PN. While in humans, meningitis is not always associated with bacteremia, PN meningitis after intravenous injection in mice usually develops following sustained and very high bacteremic titers. High grade bacteremia, however, is known to favor inflammation and BBB permeabilization, thereby increasing PN translocation across the BBB and associated damages. Therefore, specific processes associated with early events of PN translocation may be blurred by overall changes in the inflammatory environment and potentially systemic dysfunction in the case of severe sepsis. Here, we report a mouse meningitis model induced by PN injection in the retro-orbital (RO) sinus. We show that, in this model, mice appear to control bacteremic levels during the first 13 h post-infection, while PN crossing of the BBB can be clearly detected by fluorescence confocal microscopy analysis of brain slices as early as 6 h post-infection. Because of the low frequency of events, however, PN translocation across brain parenchymal vessels at early time points requires a rigorous and systematic examination of the brain volume.
Neuroscience
Conditional Gene Editing in Presynaptic Extinction-ensemble Cells via the CRISPR-SaCas9 System
The CRISPR-Cas9 enables efficient gene editing in various cell types, including post-mitotic neurons. However, neuronal ensembles in the same brain region can still be functionally or anatomically different, and such heterogeneity requires gene editing in specific neuronal populations. We recently developed a CRISPR-SaCas9 system-based technique. Combined with activity-dependent cell-labeling methods and anterograde/retrograde adeno-associated virus (AAV) vectors, this technique achieves function- and projection-specific gene editing in the mammalian brain. We showed that perturbing cbp (CREB-binding protein) in extinction-ensemble neurons among amygdala-projecting infralimbic cortex (IL) cells impaired fear extinction learning, demonstrating the high efficiency in regulation of extinction learning with CRISPR-Cas9. Here, we describe a detailed protocol of gene perturbation in presynaptic extinction-ensemble neurons in adult rats, including gRNA design, gRNA evaluation in vitro, stereotaxic AAV injection, and contextual fear conditioning. The high specificity and efficiency of projection- and function-specific CRISPR-SaCas9 system can be widely applied in neural circuitry studies.
Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster
To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and their targets RNAs, is of fundamental importance to understand the dynamic organization of the nervous system in health and disease. Nevertheless, this task has remained elusive due to the lack of specific protocols and experimental systems that would allow the combination of biochemical analysis with in vivo functional genetics. In this manuscript, we describe a trustworthy and detailed methodology to establish the molecular organization and intracellular function of RBPs/RNA multimeric complexes in a cell type-defined manner by using the powerful GAL4/UAS system for gene expression in Drosophila melanogaster.Graphic abstract: Immunoprecipitation for protein-RNA interaction in Drosophila.
Plant Science
Split-luciferase Complementation Imaging Assay to Study Protein-protein Interactions in Nicotiana benthamiana
The experimental identification of protein-protein interactions (PPIs) is critical to understand protein function. Thus, a plethora of sensitive and versatile approaches have been developed to detect PPIs in vitro or in vivo, such as protein pull-down, yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP), and bimolecular fluorescence complementation (BiFC) assays. The recently established split-luciferase complementation (Split-LUC) imaging assay has several advantages compared to other approaches to detect PPIs in planta: it is a relatively simple and fast method to detect PPIs in vivo; the results are quantitative, with high sensitivity and low background; it measures dynamic PPIs in real-time; and it requires limited experimental materials and instrumentation. In this assay, the amino-terminal and carboxyl-terminal halves of the luciferase enzyme are fused to two proteins of interest (POIs), respectively; the luciferase protein is reconstituted when two POIs interact with each other, giving rise to a measurable activity. Here, we describe a protocol for the Split-LUC imaging assay using a pair of modified gateway-compatible vectors upon Agrobacterium-mediated transient expression in Nicotiana benthamiana. With this setup, we have successfully confirmed a series of interactions among virus-plant proteins, virus-virus proteins, plant-plant proteins, or bacteria-plant proteins in N. benthamiana.
Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing
Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic’s Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.
Stem Cell
A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells
Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient.Graphic abstract: Figure 1. Satellite cell isolation using the ice-cold treatment method.
Systems Biology
TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs
High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and processivity, to convert RNAs into cDNAs for sequencing. Here, we describe an RNA-seq protocol using Thermostable Group II Intron Reverse Transcriptases (TGIRTs), which have high fidelity, processivity, and strand-displacement activity, as well as a proficient template-switching activity that enables efficient and seamless RNA-seq adapter addition. By combining these activities, TGIRT-seq enables the simultaneous profiling of all RNA biotypes from small amounts of starting material, with superior RNA-seq metrics, and unprecedented ability to sequence structured RNAs. The TGIRT-seq protocol for Illumina sequencing consists of three steps: (i) addition of a 3' RNA-seq adapter, coupled to the initiation of cDNA synthesis at the 3' end of a target RNA, via template switching from a synthetic adapter RNA/DNA starter duplex; (ii) addition of a 5' RNA-seq adapter, by using thermostable 5' App DNA/RNA ligase to ligate an adapter oligonucleotide to the 3' end of the completed cDNA; (iii) minimal PCR amplification, to add capture sites and indices for Illumina sequencing. TGIRT-seq for the Illumina sequencing platform has been used for comprehensive profiling of coding and non-coding RNAs in ribodepleted, chemically fragmented cellular RNAs, and for the analysis of intact (non-chemically fragmented) cellular, extracellular vesicle (EV), and plasma RNAs, where it yields continuous full-length end-to-end sequences of structured small non-coding RNAs (sncRNAs), including tRNAs, snoRNAs, snRNAs, pre-miRNAs, and full-length excised linear intron (FLEXI) RNAs.Graphic abstract: Figure 1. Overview of the TGIRT-seq protocol for Illumina sequencing. Major steps are: (1) Template switching from a synthetic R2 RNA/R2R DNA starter duplex with a 1-nt 3' DNA overhang (a mixture of A, C, G, and T residues, denoted N) that base pairs to the 3' nucleotide of a target RNA, and upon initiating reverse transcription by adding dNTPs, seamlessly links an R2R adapter to the 5' end of the resulting cDNA; (2) Ligation of an R1R adapter to the 3' end of the completed cDNA; and (3) Minimal PCR amplification with primers that add Illumina capture sites (P5 and P7) and barcode sequences (indices 5 and 7). The index 7 barcode is required, while the index 5 barcode is optional, to provide unique dual indices (UDIs).