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Past Issue in 2016
Volume: 6, Issue: 22
Biochemistry
Ionization Properties of Phospholipids Determined by Zeta Potential Measurements
Biological membranes are vital for diverse cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. The surface charge of the membrane bilayer plays a critical role in these myriad processes. For most biomembranes, the surface charge of anionic phospholipids contributes to the negative surface charge density within the interfacial region of the bilayer. To quantify surface charge, it is essential to understand the proton dissociation behavior of the titratable headgroups within such lipids. We describe a protocol that uses model membranes for electrokinetic zeta potential measurements coupled with data analysis using Gouy-Chapman-Stern formalism to determine the pKa value of the component lipids. A detailed example is provided for homogeneous bilayers composed of the monoanionic lipid phosphatidylglycerol. This approach can be adapted for the measurement of bilayers with a heterogeneous lipid combination, as well as for lipids with multiple titratable sites in the headgroup (e.g., cardiolipin).
Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes
Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by perturbing agents. A perturbing agent is incubated with model membranes existing as multilamellar vesicles (MLVs) and subsequently, the remaining MLVs and the small lipid/perturbing agent complexes resulting from the extraction are isolated and analysed to assess the extent and the specificity of the lipid extraction.
![Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites](https://en-cdn.bio-protocol.org/imageup/arcimg/20161108094711249.jpg?t=1758534059)
Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites
The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [3H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are incubated with radiolabelled [3H-methyl]-choline in medium for 15 min (pulse). The [3H-methyl]-choline is then rapidly removed and incubation continued in the presence of non-radioactive medium (chase). Cellular distribution of [3H-methyl] is then determined by cell fractionation and measurement of radioactivity in the lipid and non-lipid cellular components.
Cancer Biology
Establishment of Patient-Derived Xenografts in Mice
Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug evaluation and can be used for the predictive results of clinical outcomes because they conserve original tumor characteristics such as heterogeneity, complexity and molecular diversity (Kopetz et al., 2012). Additionally, PDX model provides the potential tool for the personalized drug therapy. In this protocol, we present methods for the establishment of PDX in mice using primary tumor tissues from patients with small cell lung cancer (SCLC).
Acid Extraction of Total Histone from Colon Cancer HCT116 Cells
Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H2O (ddH2O). Here we present a detailed histone acid extraction assay in our laboratory using a colon cancer cell line, HCT116, as a model.
Determining the Influence of Small Molecules on Hypoxic Prostate Cancer Cell (DU-145) Viability Using Automated Cell Counting and a Cell Harvesting Protocol
Cell viability assays are an essential aspect of most cancer studies, however they usually require a considerable labor and time input. Here, instead of using the conventional microscopy and hemocytometer cell counting approach, we developed a cell harvesting protocol and combined it with the automated Countess Automated Cell Counter to generate cell viability data. We investigated the effects of dihydroxylated bile acids on the cell viability of prostate cancer cells grown under hypoxic conditions. We observed that for all conditions, cell viability was relatively unchanged, suggesting these molecules had little or no impact on cell viability. The combination of the automated approach and the cell harvesting protocol means this assay is i) easy to perform, ii) extremely reproducible and iii) it complements more conventional cancer assay data such as invasion, migration and adhesion.
Cell Biology
Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)
The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.
Immunology
Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.
Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads
Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).
Determination of H2O2 Generation by pHPA Assay
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2 release from cells. This assay can measure H2O2 release from whole cells, mitochondria, or the NADPH oxidase.
Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).
Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.
Microbiology
Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome
Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.
Transformation of Thermus Species by Natural Competence
Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for transformation of both plasmids and linearized genomic DNA, of which the latter can be employed as an alternative method to electroporation to introduce mutations or to generate gene deletions in Thermus isolates, for instance.
Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli
The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA is regulated by the two-component system PhoP/PhoQ (Soncini et al., 1996; Kato et al., 1999). The membrane bound histidine kinase, PhoQ, senses low Mg2+ concentration in the periplasmic space and phosphorylates its cognate response regulator, PhoP, which initiates mgtA transcription (Groisman et al., 2013). MgtA is targeted to the plasma membrane and facilitate the bacterial survival under low Mg2+ condition, by importing Mg2+ into the cytoplasm. The MgtA homolog in petunia (PH1) is found in the vacuolar membrane and involved with the coloration of the flower petals (Faraco et al., 2014). As a first step towards understanding the molecular details of MgtA Mg2+ transport, we describe a detailed protocol for the purification of E. coli MgtA that can be used for biochemical and biophysical studies. Recombinant E. coli MgtA with hexa histidine tag at the N-terminus was cloned from E. coli DH5α and over expressed in the E. coli C43(DE3) by fermentation to an OD > 6. Cell lysis was performed in a high pressure homogenizer and the membranes were isolated by ultracentrifugation. Membrane proteins were solubilized with the detergent dodecyl-β-D maltoside. MgtA was purified by affinity and size exclusion chromatography. Final yields of purified MgtA reach ~1 mg MgtA per 3 g of wet cell pellet.
Cell-to-cell DNA Transfer among Thermus Species
The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the selection and quantification of the transfer frequencies. Briefly, liquid cultures of both mates are mixed and laid onto a nitrocellulose filter on a TB plate. After incubation at 60 °C, filters are resuspended upon selective plating. The contribution of DNA uptake by transformation is abolished by the addition of DNase I to the mix. This protocol can be used for the transfer of large DNA fragments (> 10 kb) to Thermus species.
Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process. Figure 1. Model for virus-induced caspase-8-dependent proteolysis of IRF3. In virus-infected cells, IRF3 can be proteolytically cleaved by caspase-8, which gets activated during infection. The cleaved IRF3 is subjected to poly-ubiquitination (Ub) and degradation by proteasome machinery. The degradation of IRF3 inhibits dsRNA-induced antiviral gene induction.
Halo Assay for Toxic Peptides and Other Compounds in Microorganisms
We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing S. cerevisiae with the target microbe and modifying growth conditions accordingly.
Uptake Assay for Radiolabeled Peptides in Yeast
We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.
Neuroscience
Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice
In order to explore the role of a specific gene/protein in the specific segment of the spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, lentivirus containing shRNA for CXCL13 were injected into the dorsal horn of the spinal lumbar enlargement segment (Jiang et al., 2016). This technique allows the study of the role of CXCL13 in the ipsilateral dorsal horn in neuropathic pain without affecting DRG or contralateral dorsal horn.
In vitro Brainstem-spinal Cord Preparation from Newborn Rat
The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).
An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties
Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.
Plant Science
Arabidopsis Seed Germination Assay with Gibberellic Acid
This assay analyzes Arabidopsis seed germination in response to gibberellic acid (GA). During seed imbibition, visible physiological changes allow precise determination of germination rate. This protocol utilizes a stereoscopic microscope to improve characterization of seed germination process.
Documentation of Floral Secretory Glands in Pleurothallidinae (Orchidaceae) Using Scanning Electron Microscopy (SEM)
A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.
Cryopreservation Protocol for Chlamydomonas reinhardtii
Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang and Li, 2016). In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step cryopreservation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate.
Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis
Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate spline analysis is a method for visualizing the change of size and shape of objects through warping or deformation of a regular mesh and can be applied to understand cell wall growth. This protocol describes the application of thin plate spline analysis to visualize the development of individual pavement cells over time.
Stem Cell
Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies
The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then isolated through culturing, filtering and magnetic separation using cell-surface antigen markers. Here we provide a comprehensive protocol on how to isolate and culture individual cell types from whole endometrial tissues for use in in vitro experiments.
Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments
Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications. This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.