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Past Issue in 2017
Volume: 7, Issue: 7
Biochemistry
Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies
Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. We also explain how to derive distance restraints for paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) studies.
Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition
Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides is a critical tool for assessing the structural composition of this class of compound. This protocol details a methodology to reproducibly determine the disaccharide composition of heparan sulfate by enzymatic depolymerisation and SAX-HPLC analysis. A complementary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).
Heparan Sulfate Identification and Characterisation: Method I. Heparan Sulfate Identification by NMR Analysis
Heparin and heparan sulfate (HS) may be purified from complex biological matrices and are often isolated in sub-milligram quantities but not unequivocally identified by spectroscopic means. This protocol details a methodology to rapidly assess the gross compositional features and approximate purity of HS by 1H nuclear magnetic resonance. A complimentary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).
Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth
We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson-Thomas et al., 2015) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method (Nader et al., 1999) which we optimized for archeological samples.
Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER)
In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.
Cell Biology
Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics
The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.
Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites
Ovarian cancer is one of the most fatal tumors in women. Due to a lack of symptoms and adequate screening methods, patients are diagnosed at advanced stages with extensive tumor burden (Jelovac and Armstrong, 2011). Interestingly, ovarian cancer metastasis is generally found within the peritoneal cavity rather than other tissues (Lengyel, 2010; Tan et al., 2006). The reason behind this tissue tropism of the peritoneal cavity remains elusive. A prominent feature of this selectivity is ascites, the accumulation of fluid within the peritoneal cavity, containing, amongst others, immune cells, tumor cells and various soluble factors that can be involved in the progression of ovarian cancer (Kipps et al., 2013). The protocol described here is used to isolate mononuclear cells from ascites to study the functionality of the immune system within the peritoneal cavity.
In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans
Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of germline microinjection of a chemical calcium indicator, worm immobilization, live imaging, and image processing that quantifies the calcium fluorescence in the oocyte region moving in the field-of-view during ovulation. This imaging protocol can also be used to image other cellular processes during in vivo fertilization in C. elegans, such as membrane fusion and cytoskeletal dynamics.
In vitro Microtubule Bundling Assay under Physiological Conditions
Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.
Developmental Biology
Assessment of Murine Retinal Function by Electroretinography
The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.
In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis
Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.
Immunology
Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized by recipient neighboring or distal cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. Exosomes facilitate intercellular communication, modulate cellular phenotype, and regulate microbial pathogenesis. We have previously shown that semen exosomes (SE) inhibit HIV-1 replication in various cell types. Here, we describe detailed protocols for characterizing SE. This protocol can be adapted or modified and used for evaluation of other extracellular vesicles of interest.
Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay
An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.
In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae
In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of immune cells on fungi and the cell wall, making them promising tools for exploring host-pathogen interactions during fungal infection.
Microbiology
RNA-dependent RNA Polymerase Assay for Hepatitis E Virus
RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.
Measurement of the Galactanase Activity of the GanB Galactanase Protein from Bacillus subtilis
The activity of the endo-β-1,4-galactanase GanB from B. subtilis on the high molecular weight β-1,4-galactan was determined quantitatively by the measurement of the increase of the reducing power or with the dyed substrate Azo-galactan. The generated degradation products were analyzed using thinlayer-chromatography (TLC) or high-performance anion-exchange chromatography (HPAEC).
ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors
The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., 2015). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm (Michard et al., 2015). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.
RNA Strand Displacement Assay for Hepatitis E Virus Helicase
The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.
Purification of N-coronafacoyl Phytotoxins from Streptomyces scabies
This procedure is used for large-scale purification of N-coronafacoyl phytotoxins that are produced by the potato common scab pathogen Streptomyces scabies. The procedure employs organic extraction of S. scabies culture supernatants under alternating basic and acidic conditions in order to preferentially isolate the phytotoxin - containing carboxylic acid fraction of the supernatant. Preparative thin layer chromatography and semi-preparative reverse phase - high performance liquid chromatography are then used to further purify the individual N-coronafacoyl phytotoxins of interest.
Molecular Biology
Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.
Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.
Neuroscience
Testing Depression in Mice: a Chronic Social Defeat Stress Model
A vast challenge within neuropsychiatric research has been the development of animal models that accurately reflect symptoms associated with affective disorders. An ethologically valid model that has been shown to be effective in studying depression is the chronic social defeat stress model. In this model, C57BL/6J mice are subjected to chronic social defeat stress induced by CD-1 aggressor mice for 10 consecutive days. Discussed here is a protocol describing the screening process of the CD-1 aggressor mice, the confrontations between the C57BL/6J and CD-1 aggressor mice, and analysis of social avoidance scores as an indication of depression-like behaviors.
Measuring Behavioral Individuality in the Acoustic Startle Behavior in Zebrafish
The objective of this protocol is to provide a detailed description for the construction and use of a behavioral apparatus, the zBox, for high-throughput behavioral measurements in larval zebrafish (Danio rerio). The zBox is used to measure behavior in multiple individuals simultaneously. Individual fish are housed in wells of multi-well plates and receive acoustic/vibration stimuli with simultaneous recording of behavior. Automated analysis of behavioral movies is performed with MATLAB scripts. This protocol was adapted from two of our previously published papers (Levitz et al., 2013; Pantoja et al., 2016). The zBox provides an easy to setup flexible platform for behavioral experiments in zebrafish larvae.
Plant Science
Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration
CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end joining (NHEJ) pathway (Wang et al., 2016). Since NHEJ is error prone, mutations are thus generated. In plants, delivery of genome editing reagents is still challenging. In this protocol, we detail the procedure of a virus-based gRNA delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE). This method offers a rapid and efficient way to deliver gRNA into plant cells, especially for those that are recalcitrant to transformation with Agrobacterium.
Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis
Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to release Pi from organophosphate compounds in the rhizosphere. The phosphate starvation-induced secreted APases can be released into the rhizosphere or retained on root surfaces (root-associated APases). In this article, we describe the protocols for analyzing root-secreted APase activity in the model plant Arabidopsis thaliana (Arabidopsis). In Arabidopsis, the activity of both root-associated APases and APases that are released into the rhizosphere can be quantified based on their ability to cleave a synthesized substrate, para-nitrophenyl-phosphate (pNPP), which releases a yellow product, para-nitrophenol (pNP) (Wang et al., 2011 and 2104). The root-associated APase activity can also be directly visualized by applying a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), to the root surface (Lloyd et al., 2001; Tomscha et al., 2004; Wang et al., 2011 and 2014) whereas the isozymes of APases that are released into rhizosphere can be profiled using an in-gel assay (Trull and Deikman, 1998; Tomscha et al., 2004; Wang et al., 2011 and 2014). The protocol for analysis of intracellular APase activity in Arabidopsis has been previously described (Vicki and William, 2013).
Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining
This protocol was developed to study protein localisation within the vascular bundles of developing tomato fruit however, it can be applied to any resin embedded plant tissue. The vascular bundle is comprised of many different cells that all have unique properties. The mature sieve elements are enucleated and contain sieve plates that comprise of callose. This method has utilised these properties of the sieve element by combining immunohistochemistry for cell wall invertase with counterstaining of aniline blue for callose, DAPI for nucleus and cell structure is shown with the final staining of the cell wall using calcofluor white. It must be noted that when following this protocol, it is vital for the sections to be flat and fixed to the slide with gelatine so cover slip removal does not move the sample section. This protocol will be applicable to all plant tissues and provides additional evidence of the protein localisation within the cell by conducting a counterstaining procedure.
Stem Cell
A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells
In glaucoma, the output neurons of the retina, the retinal ganglion cells (RGCs), progressively degenerate, leading to irreversible blindness (Ahram et al., 2015). The ex vivo stem cell method to replace degenerated RGCs remains a potentially viable approach (Levin et al., 2004). However, the success of the approach depends upon the ability of the de novo generated RGCs to connect over the long distance with specific targets in the central visual pathway. Here, we describe a protocol to examine the target specificity of the de novo generated RGCs using a co-culture approach where the RGCs neurites are allowed to choose between specific (superior colliculus; SC) and non-specific (inferior colliculus; IC) tectal targets.