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Past Issue in 2018
Volume: 8, Issue: 13
Biochemistry
Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
Rat tail collagen solutions have been used as polymerizable in vitro three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the in vivo physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen. Here we detail how to generate 3D collagen gels labeled with a fluorescent dye. Furthermore, we go through the steps required to reproducibly generate bright collagen hydrogels that are suitable for live cell 3D imaging techniques.
Assessment of Uptake and Biodistribution of Radiolabeled Cholesterol in Mice Using Gavaged Recombinant Triglyceride-rich Lipoprotein Particles (rTRL)
The here described method can be used to estimate the uptake of orally provided cholesterol in mice. Briefly, mice are gavaged with radiolabeled cholesterol and 4 h later, organ distribution of the radiolabel is determined by liquid scintillation counting. The method has been applied successfully to determine dietary cholesterol handling of mice housed at different ambient temperatures
Cancer Biology
An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist
One fundamental property of the TNR receptor (TNFR) family relates to how ‘signal quality’ (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., agonistic anti-CD40 antibody), induces high level of pro-inflammatory cytokine secretion and causes extensive cell death (apoptosis) in malignant (but not normal) epithelial cells. In this article, we describe a co-culture system for the activation of CD40 by mCD40L and subsequent detection of various features of apoptosis (including cell membrane permeabilisation, DNA fragmentation, caspase activation) as well as detection of intracellular mediators of cell death (including adaptor proteins, pro-apoptotic kinases and reactive oxygen species, ROS).
Cell Biology
FRAP: A Powerful Method to Evaluate Membrane Fluidity in Caenorhabditis elegans
FRAP (Fluorescence Recovery After Photobleaching) is probably the most direct method to investigate the dynamics of molecules in living cells. Here, we describe FRAP to quantify membrane fluidity in C. elegans. Using FRAP, we have shown that cold, glucose and exogenous saturated fatty acids can decrease the fluidity of cellular membranes in certain mutants.
Developmental Biology
Mouse Mammary Gland Whole Mount Preparation and Analysis
The mammary gland undergoes extensive remodeling during pregnancy and is also subject to neoplastic processes both of which result in histological changes of the gland epithelial structure. Since the mammary tree is a complex three-dimensional structure a method is needed that provides an overview of the entire gland. Whole mounts provide this information, are inexpensive and do not require specialized equipment. This protocol describes mammary gland isolation, whole mount preparation and analysis. Mammary gland tissue, which is removed postmortem, is stained with Carmine Alum, a nuclear stain, allowing detection of epithelial structures embedded in the adipose tissue of the mammary fat pad. Stained mammary glands are imaged by light microscopy or embedded and sectioned for histological examination. Image analysis software such as Image J can be used to quantify extensity of branching complexity, epithelial structure remodeling or hyperplastic changes.
Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.
Microbiology
Bacterial Microcolonies in Gel Beads for High-throughput Screening
High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies inside the beads. After isolation of the gel beads from the emulsion and their sorting by fluorescence activated cell sorting (FACS), the bacteria are recovered from the gel beads and are then ready for a further round of sorting, mutagenesis or analysis. In order to sort by FACS, this protocol requires a fluorescent readout, such as the expression of a fluorescent reporter protein. Measuring the average fluorescent signals of microcolonies reduces the influence of high phenotypic cell-to-cell variability and increases the sensitivity compared to the sorting of single cells. We applied this method to sort a pBAD promoter library at ON and OFF states (Duarte et al., 2017).
Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines
The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.
Induction of Natural Competence in Genetically-modified Lactococcus lactis
Natural competence can be activated in Lactoccocus lactis subsp lactis and cremoris upon overexpression of ComX, a master regulator of bacterial competence. Herein, we demonstrate a method to activate bacterial competence by regulating the expression of the comX gene by using a nisin-inducible promoter in an L. lactis strain harboring either a chromosomal or plasmid-encoded copy of nisRK. Addition of moderate concentrations of the inducer nisin resulted in concomitant moderate levels of ComX, which led to an optimal transformation rate (1.0 x 10-6 transformants/total cell number/g plasmid DNA). Here, a detailed description of the optimized protocol for competence induction is presented.
Neuroscience
Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure culture of CGN from postnatal rats/mice and an easy procedure for their intoxication with CNT. We also illustrate handy methods to evaluate CNT activity and their inhibition.
Brain Tissue Culture of Per2::Luciferase Transgenic Mice for ex vivo Bioluminescence
In circadian research, it is essential to be able to track a biological rhythm for several days with the minimum perturbation for the organisms or tissues. The use of transgenic mice lines, in which the luciferase reporter is coupled to a molecular clock protein (here PERIOD2), gives us the opportunity to follow the circadian activity in different tissues or even single clock cells for days without manipulation. This method creates sections using a mouse brain matrix, which allows us to obtain several brain samples quickly at a single time point.
Plant Science
An Optimized CTAB Method for Genomic DNA Extraction from Freshly-picked Pinnae of Fern, Adiantum capillus-veneris L.
As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris L. In 500 μl CTAB solution, the recommended amount of pinnae is about 10-20 mg (2-3 pieces). The condition of the pinnae must be instantly-picked from a plant cultivated in a suitable environment. With these factors under control, it is highly reproducible to get the high-quality gDNA with low degradation rate
Quantification of Starch in Guard Cells of Arabidopsis thaliana
In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.
Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE
The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from Arabidopsis thaliana.
Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds
Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).
A Modified Approach for Axenic Cultivation of Spores of Fern Adiantum capillus-veneris L. with High Germination Rate
Spores are the primary way of spread and reproduction for ferns, a clade of seed-free vascular plants. However, no detailed protocol for ferns spore cultivation has been reported yet. Here we provide a modified approach for axenic cultivation of fern Adiantum capillus-veneris L., based on Cao’s and Li’s method (Cao, et al., 2010; Li, et al., 2013).Our approach can be briefly divided into four steps: 1) collect spores; 2) sterilize the spores with 5% sodium hypochlorite solution and wash twice; 3) incubate the spores in liquid Knop’s medium in the dark for five days; 4) cultivate the spores on Knop's plate medium. To increase the germination rate, we constrain the sterilization time under 25 min and add dark treatment step after spore sterilization. After these modifications, the germination rate raises from 2% to 25%.