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Past Issue in 2021
Volume: 11, Issue: 7
Biochemistry
Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication (Liu et al., 2019). The procedure may be adapted for functional and structural studies.
Biophysics
FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.
Developing Biohybrid Robotic Jellyfish (Aurelia aurita) for Free-swimming Tests in the Laboratory and in the Field
Biohybrid robotics is a growing field that incorporates both live tissues and engineered materials to build robots that address current limitations in robots, including high power consumption and low damage tolerance. One approach is to use microelectronics to enhance whole organisms, which has previously been achieved to control the locomotion of insects. However, the robotic control of jellyfish swimming offers additional advantages, with the potential to become a new ocean monitoring tool in conjunction with existing technologies. Here, we delineate protocols to build a self-contained swim controller using commercially available microelectronics, embed the device into live jellyfish, and calculate vertical swimming speeds in both laboratory conditions and coastal waters. Using these methods, we previously demonstrated enhanced swimming speeds up to threefold, compared to natural jellyfish swimming, in laboratory and in situ experiments. These results offered insights into both designing low-power robots and probing the structure-function of basal organisms. Future iterations of these biohybrid robotic jellyfish could be used for practical applications in ocean monitoring.
Cancer Biology
Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP)
We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.
Developmental Biology
A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote (Rahman et al., 2020).
Analysis of TORC1-body Formation in Budding Yeast
The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.
Immunology
Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response
Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a robust model for activation of innate immunity by microinjecting bacterial particles or live bacteria into larval zebrafish, unlike previous studies which largely restricted such manipulations to embryonic stages of zebrafish. The ability to introduce stimuli locally or systemically at larval stages provides significant advantages to examine host response in more mature tissues as well as the possibility to interrogate adaptive immunity at older larval stages. This protocol describes two distinct modes of microinjection to introduce lipopolysaccharide (LPS) or bacteria into the living larval zebrafish: one localized to the brain, and another into the bloodstream via the caudal vein plexus.Graphic abstract: Schematic shows the two distinct modes of larval zebrafish microinjection, either in the brain parenchyma or in the blood stream intravenously. Reagents introduced into the zebrafish to assess immune response are depicted in the “injection components” as described in the protocol.
A Potent Vaccine Delivery System
Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery system that is fully defined, biodegradable, and non-toxic. The system is produced by conjugation of polyleucine to peptide antigen, followed by self-assembly of the conjugate into nanoparticles. The protocol includes solid-phase peptide synthesis of the vaccine conjugate, purification, self-assembly and physicochemical characterization of the product. Overall, this protocol describes, in detail, the production of a well-defined and effective self-adjuvanting delivery system for peptide antigens, along with tips for troubleshooting.
Using the Cecal Ligation and Puncture Model of Sepsis to Induce Rats to Multiple Organ Dysfunction
Sepsis is a dysregulated hyperinflammatory disease caused by infection. Sepsis leads to multiple organ dysfunction syndrome (MODS), which is associated with high rates of mortality. The cecal ligation and puncture (CLP) model has been widely used in animals and has become the gold-standard method of replicating features of sepsis in humans. Despite several studies and modified CLP protocols, there are still open questions regarding the multifactorial determinants of its reproducibility and medical significance. In our protocol, which is also aimed at mimicking the sepsis observed in clinical practice, male Wistar rats are submitted to CLP with adequate fluid resuscitation (0.15 M NaCl, 25 ml/kg BW i.p.) immediately after surgery. At 6 h after CLP, additional fluid therapy (0.15 M NaCl, 25 ml/kg BW s.c.) and antibiotic therapy with imipenem-cilastatin (single dose of 14 mg/kg BW s.c.) are administered. The timing of the fluid and antibiotic therapy correspond to the initial care given when patients are admitted to the intensive care unit. This model of sepsis provides a useful platform for simulating human sepsis and could lay the groundwork for the development of new treatments.
Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer
Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That’s why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.
Molecular Biology
Reconstitution of Chromatin by Stepwise Salt Dialysis
Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for a wide range of applications. Here, we describe simple and straightforward protocols for the reconstitution of chromatin by stepwise salt dialysis and the analysis of the chromatin by the micrococcal nuclease (MNase) digestion assay. Chromatin that is reconstituted with this method can be used for efficient homology-directed repair (HDR)-mediated gene edited with the CRISPR-Cas9 system as well as for biochemical studies of chromatin dynamics and function.
Neuroscience
Cranioplastic Surgery and Acclimation Training for Awake Mouse fMRI
MRI is a promising tool for translational research to link brain function and structure in animal models of disease to patients with neuropsychiatric disorders. However, given that mouse functional MRI (fMRI) typically relies on anesthetics to suppress head motion and physiological noise, it has been difficult to directly compare brain fMRI in anesthetized mice with that in conscious patients. Here, we developed a new system to acquire fMRI in awake mice, which includes a head positioner and dedicated radio frequency coil. The system was used to investigate functional brain networks in conscious mice, with the goal of enabling future studies to bridge fMRI of disease model animals with human fMRI. Cranioplastic surgery was performed to affix the head mount and the cupped-hand handling method was performed to minimize stress during MRI scanning. Here we describe the new mouse fMRI system, cranioplastic surgery and acclimation protocol.Graphic abstract:Awake fMRI system to investigate the neuronal activity in awaked mice.
Imaging Microtubules in vitro at High Resolution while Preserving their Structure
Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can then be observed using a confocal microscope with an Airyscan module for higher resolution. This protocol allows the imaging of microtubules that have retained their original three-dimensional shape and is compatible with high-resolution immunofluorescence detection.
Plant Science
Measurements of Root Colonized Bacteria Species
Root-associated bacteria are able to influence plant fitness and vigor. A key step in understanding the belowground plant-bacteria interactions is to quantify root colonization by the bacteria of interest. Probably, genetic engineering with fluorescence markers is the most powerful way to monitor bacterial strains in plant. However, this could have some collateral problems and some strains can be challenging to label. In this sense, bacterial inoculation under properly controlled conditions can enable reliable and reproducible quantification of natural bacterial strains. In this protocol, we describe a detailed procedure for quantification of root-associated bacteria. This method applies non-aggressive samples processed with morphological identification and PCR-based genetic fingerprinting. This easy-to-follow protocol is suitable for studying bacterial colonization of plants grown either in artificial medium or in soil.
In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants.
A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.