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Past Issue in 2017
Volume: 7, Issue: 2
Biochemistry
Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model
This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in embryonated eggs, larval stages and in the adults’ survival. As most current anthelmintics are not effective against all nematode life stages, this protocol can contribute to the identification of new alternatives to helminthic infections (Sant’Anna et al., 2016).
Cancer Biology
Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.
In vitro Dephosphorylation Assay of c-Myc
This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases.
Developmental Biology
P-body and Stress Granule Quantification in Caenorhabditis elegans
Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules is Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans.
Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin
Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.
Immunology
Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates the Ficoll solution from the blood to be drawn. Once blood is drawn into CPTs, they can be centrifuged to separate the PBMC, then shipped (if desired) to a processing lab. The processing lab removes the PBMC from the upper compartment of the tube (above the gel plug), washes the PBMC, and can cryopreserve them using DMSO-containing media, as detailed in this protocol.
Virus Binding and Internalization Assay for Adeno-associated Virus
The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based method to determine the amount of vector bound to the cell surface and the amount of subsequent virus internalization based on viral genome quantification. This protocol is optimized for studying AAV. Nevertheless, it can serve as a backbone for studying other viruses with careful modification.
Microbiology
Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and NdeI. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and NdeI) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a Mycobacterium tuberculosis protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/NdeI aggregation.Figure 1. Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I1, I2) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.
Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells
This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol allows the production of reproducible results in a relatively short time for Sinorhizobium meliloti 1021 cells grown both in exponential and stationary phases. The results here presented show that the S. meliloti cells deriving from exponential-phase cultures had increased cell redox potential as compared to the ones deriving from stationary-phase cultures. This means that under exponential growth conditions the S. meliloti cells generate higher amount of reducing equivalents needed for TTC reduction.
Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor
The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.
Neuroscience
An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo
Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord’s highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord. Here, we describe a procedure for preparing acute slices from mouse spinal cord (Garré et al., 2016). This preparation should allow further understanding of how glial cells in spinal cord respond acutely to various inflammatory challenges.
Plant Science
Nitrate Assay for Plant Tissues
Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement of nitrate in plant tissues.
Pseudomonas syringae Flood-inoculation Method in Arabidopsis
Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis.
Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) transmembrane receptor-like protein. Data are presented showing reconstitution of abscisic acid activation of the SLAC1 anion channel by the ‘monomeric’ ABA (abscisic acid) receptor RCAR1/PYL9 (PYRABACT INRESISTANCE1 [PYR1]/PYR1-LIKE [PYL]/REGULATORYCOMPONENTS OF ABA RECEPTORS [RCAR]) by co-expressing four components of the abscisic acid signaling core. This protocol is also suitable for studying other ion channel functions and regulation mechanisms, as well as transporter proteins.
Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves
Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases of sugarcane. Dissemination of X. albilineans is mainly ensured by contaminated harvesting tools and infected stalk cuttings. However, some strains of this pathogen are transmitted by aerial means and are able to survive as epiphytes on the sugarcane phyllosphere before entering the leaves and causing disease. Here we present a protocol to estimate the capacity of attachment of X. albilineans to sugarcane leaves. Tissue-cultured sugarcane plantlets were immersed in a bacterial suspension of X. albilineans and leaf attachment of X. albilineans was determined by two methods: leaf imprinting (semi-quantitative method) and leaf washing/homogenization (quantitative method). These methods are important tools for evaluating pathogenicity of strains/mutants of the sugarcane leaf scald pathogen.
In vitro Floral Induction of Cuscuta reflexa
Floral initiation and development in the angiosperms is the essential step on which the yield of the plant depends. Sometimes external climate or any abiotic stress hinders the floral initiation and ultimately affect the plant yield. Hence, in vitro floral induction and development can help to overcome the external climatic factor. Furthermore, the protocol for in vitro floral induction in the parasitic angiosperm like Cuscuta reflexa has not been reported yet. We have standardized the protocol for floral induction in the parasitic plant Cuscuta reflexa. In this study it is established that MMS (modified Murashige Skoog) media supplemented with 2 mg L-1 NAA (naphthalene acetic acid, plant growth regulator) and 2 mg L-1 2,4-D (2,4-dichlorophenoxy acetic acid, plant growth regulator) supported floral induction after shooting in vitro. Furthermore, we found that MMS media supplemented with 2 mg L-1 2,4-D rapidly induced floral buds directly from the nodal explants without any shoot elongation. This protocol will help the researcher to induce flower in vitro in the other angiosperm plants along with Cuscuta reflexa.