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Past Issue in 2018
Volume: 8, Issue: 16
Biochemistry
Modifying Styrene-maleic Acid Co-polymer for Studying Lipid Nanodiscs by Direct Fluorescent Labeling
This protocol was developed to functionalize styrene maleic acid (SMA) by direct fluorescent labeling in an easy way, accessible to biochemistry laboratories. This novel method is based on the coupling of carboxylic acids to primary amines using a carbodiimide, a reaction commonly used for protein chemistry. The procedure uses the hydrolyzed styrene-maleic acid copolymer and occurs entirely in aqueous solution with mild conditions compatible with many biomolecules.
Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in MCF10A mammary epithelial and Caki-1 renal cancer epithelial cells. Using two detergent extractions, cellular FN is separated into detergent insoluble or fibril incorporated FN and soluble FN or unincorporated fractions. To determine whether fibrillogenesis utilizes a recycled pool of FN, we have used a Biotin labeled FN (FN-Biotin) recycling assay, that has been modified from a previous study. Using a combination of the recycling assay and deoxycholate fractionation methods, one can quantitatively demonstrate the extent of fibrillogenesis in cells under different experimental conditions and determine the source of FN for fibrillogenesis.
Immunology
Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry
Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. This methodology is not amenable to the combinations of multiple markers necessary to unequivocally define discrete immune cell populations. We have developed a multi-parameter, flow cytometric-based approach that addresses the need for panels of cell-specific markers in the identification of immune cell populations, allowing both the accurate detection and quantitation of leukocyte subpopulations from a single, clinical kidney biopsy specimen. In this approach, fresh human kidney tissue is dissociated into a single cell suspension followed by antibody-labeling and flow cytometric-based acquisition and analysis. This novel technique provides a major step forward in identifying and enumerating immune cell subpopulations in human kidney disease and is a powerful platform to complement traditional histopathological examinations of clinical kidney biopsies.
Microbiology
Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes
Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.
CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.
Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using plasmid-based copies of the biosynthetic genes as selective markers. Previous work on gene knockouts in P. tricornutum used biolistic transformation to deliver CRISPR-Cas9 system into P. tricornutum. Biolistic transformation of non-replicating plasmids can cause undesired damage to P. tricornutum due to random integration of the transformed DNA into the genome. Subsequent curing of edited cells to prevent long-term overexpression of Cas9 is very difficult as there is currently no method to excise integrated plasmids. This protocol adapts a new method to deliver the Cas9 or TevCas9 system into P. tricornutum via conjugation of plasmids from a bacterial donor cell. The process involves: 1) design and insertion of a guideRNA targeting the P. tricornutum urease gene into a TevCas9 expression plasmid that also encodes a conjugative origin of transfer, 2) installation of this plasmid in Escherichia coli containing a plasmid (pTA-Mob) containing the conjugative machinery, 3) transfer of the TevCas9 expression plasmid into P. tricornutum by conjugation, 4) screening of ex-conjugants for urease knockouts using T7 Endonuclease I and phenotypic screening, and 5) curing of the plasmid from edited cells.
Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions
Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for observing time-resolved, multi-species interactions at single-cell resolution. The protocol involves growing bacterial cells in a near monolayer in a microfluidic device. As a demonstration, we describe in particular observing the dynamic interactions between E. coli and Acinetobacter baylyi. In this case, the protocol is capable of observing both contact-dependent lysis of E. coli by A. baylyi via the Type VI Secretion System (T6SS) and subsequent functional horizontal gene transfer (HGT) of genes from E. coli to A. baylyi.
In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals
We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).
Assessment of Caenorhabditis elegans Competitive Fitness in the Presence of a Bacterial Parasite
Accurate measurements of an organism’s fitness are crucial for measuring evolutionary change. Methods of fitness measurement are most accurate when incorporating an individual’s survival and fecundity, as well as accounting for any ecological interactions or environmental effects experienced by the organism. Here, we describe a protocol for measuring the relative mean fitness of Caenorhabditis elegans populations, or strains, through an assay that accounts for individual survival, fecundity, and intraspecific competitive ability in the presence of a bacterial parasite. In this competitive fitness assay nematodes from a focal population or strain are mixed with a GFP-marked tester strain in equal proportions, the mixture of nematodes are then exposed to a parasite, and the relative competitive fitness of the focal strain is determined by measuring the change in the ratio of focal nematodes to GFP-marked nematodes after one generation. Specifically, this protocol can be implemented to measure changes in nematode host fitness after experimental evolution by determining the relative competitive fitness of evolved versus ancestral nematode populations.
Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis. A library of viral genomes, each containing a single randomly placed small insertion, is selected by passaging in cell culture and the insertion sites can be identified using Next Generation Sequencing (NGS). Here we describe a protocol for transposon mutagenesis of the 16681 strain of dengue virus, serotype 2. Mutant dengue virus libraries containing short randomly placed insertions are passaged through mammalian cells and insertions are mapped by NGS of the viable progeny. The protocol is divided into four stages: transposon mutagenesis of a dengue cDNA clone, viral genome transfection into permissive cells, isolation of viral progeny genomes, and sequencing library preparation.
Neuroscience
Studying the Role of Microglia in Neurodegeneration and Axonal Regeneration in the Murine Visual System
Microglia reside in the central nervous system (CNS) and are involved in the maintenance of the physiologic state. They constantly survey their environment for pathologic alterations associated with injury or diseases. For decades, researchers have investigated the role of microglia under different pathologic conditions, using approaches aiming to inhibit or eliminate these phagocytic cells. However, until recently, methods have failed to achieve complete depletion. Moreover, treatments often affected other cells, making unequivocal conclusions from these studies difficult. Recently, we have shown that inhibition of colony stimulating factor 1 receptor (CSF1R) by oral treatment with PLX5622 containing chow enables complete depletion of retinal microglia and almost complete microglia depletion in the optic nerve without affecting peripheral macrophages or other cells. Using this approach, we investigated the role of microglia in neuroprotection in the retina and axon regeneration in the injured optic nerve under different conditions. Thus, this efficient, reliable and easy to use protocol presented here will enable researchers to unequivocally study the contribution of microglia on neurodegeneration and axon regeneration. This protocol can be also easily expanded to other paradigms of acute and chronic injury or diseases in the visual system.
Trace Fear Conditioning: Procedure for Assessing Complex Hippocampal Function in Mice
The trace fear conditioning protocol is designed to measure hippocampal function in mice. The protocol includes a neutral conditioned stimulus (tone) and an aversive unconditioned stimulus (shock), separated in time by a trace interval. The trace interval between the tone and the shock critically involves the hippocampus and could be used to evaluate hippocampal-dependent learning and memory. In this protocol, we presented mice with five pairings of tone and shock separated by a 20 sec trace interval. Freezing was measured 24 h after conditioning to evaluate contextual memory by placing mice in the conditioned chamber. In addition, 48 h after conditioning, freezing was measured in a dark chamber, which served as a different context. This method enables precise detection of hippocampal-dependent learning and memory following pharmacological and genetic manipulations that impair or enhance hippocampal function.
Image-Based Analysis of Mitochondrial Area and Counting from Adult Mouse Dopaminergic Neurites
Mitochondria form dynamic cytoplasmic networks which undergo morphological changes in order to adapt to cellular stresses and signals. These changes can include alterations in size and number within a given cell. Analysis of the whole network can be a useful metric to assess overall mitochondrial health, particularly in neurons, which are highly sensitive to mitochondrial dysfunction. Here we describe a method which combines immunofluorescence and computerized image analysis to measure mitochondrial morphology (quantification of number, density, and area) in dopaminergic neurites of mice expressing mitochondrially-targeted eYFP.
Plant Science
Pollen Germination and Pollen Tube Growth of Arabidopsis thaliana: In vitro and Semi in vivo Methods
Studies of pollen germination and post-germination development are not only essential for understanding plant reproduction but also are an excellent model system for tip-based growth. Here we describe easy, reproducible methods for germination and growth of pollen from the model plant Arabidopsis thaliana in artificial conditions. Our growth system can be used both for pollen placed directly on this artificial substrate as well as for the so-called ‘semi in vivo’ method. This is where a pistil is cut shortly after hand-pollination and the pollen tubes grow through the plant tissue and emerge from the cut end onto the surface of the artificial medium.
An Inexpensive and Comprehensive Method to Examine and Quantify Field Insect Community Influenced by Host Plant Olfactory Cues
Insect pollinators, herbivores and their natural enemies use olfactory cues emitted by their host plants to locate them. In insect-plant ecology, understanding the mechanisms underlying these interactions are of critical importance, as this bio-communication has both ecological and agricultural applications. However, the first step in such research is to identify and quantify the insect community associated with the plant/s species of interest. Traditionally, this has been accomplished by a variety of insect trapping methods, either using pitfall traps, or sticky traps, or sweep nets in field. The data collected from these traps tend to be incomplete, and also damage the specimens, making them unusable for any taxonomic purposes. This protocol derives ideas from these traditional traps and use a combination of three easily made inexpensive modified traps that conceals the host plant, but allows the plant volatiles to pass through as olfactory cues. These traps are economical, can be made to fit with most plant sizes, and are also reusable. Collectively, these traps will provide a solid estimate (quantifiable) of all associated community of arthropods that can also be stored for future studies.
Stem Cell
Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates
The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding 96-well plate.Spheroids of hMSCs cultured in low-binding 96-well plates can be used to study the basic biology of the cells and to generate conditioned media or spheroids to be used in transplantation therapeutic approaches. These MSCs or their secretome can be used as a regenerative therapy and for tissue repair across multiple disease areas, including neurodegeneration.In comparison to other methods (hanging drop, use of gels or biomaterials, magnetic levitation, etc.), the method described here is simple and affordable with no need to use specialized equipment, expensive materials or complex reagents.
Systems Biology
Quantitative ChIP-seq by Adding Spike-in from Another Species
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues.
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