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Past Issue in 2014
Volume: 4, Issue: 16
Cancer Biology
Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment
Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific T cell response within the tumor infiltrating lymphocytes is a more accurate indicator or vaccine efficacy. However, multi-parameter flow cytometric analysis of T cells isolated from tumor tissue can be quite challenging due to the over-whelming number of tumor cells present in relation to the tumor infiltrating lymphocytes (TIL) and to problems associated to the large and adhesive nature of many tumor cell. Here we take advantage of a pre-flow separation of CD45+ leukocytes from the tumor tissue using the MACS magnetic cell sorting system, resulting in a much cleaner cell preparation with which to proceed to flow cytometric staining and analysis.
Vaccine-induced Cytokine Production Detected by Luminex Multiplex Analysis
Different vaccine and adjuvant combinations are known to rapidly induce antigen presenting cell (APC) maturation and pro-inflammatory cytokine and production, which in turn play an important role in the priming of antigen-specific T cells. Measuring cytokine production systemically in the serum fails to detect localized responses in the lymph nodes draining a subcutaneous immunization site. On the other hand, stimulating APC with vaccine formulations in vitro lacks the complexity of the lymph node microenvironment and the presence of other in vivo factors. Here we analyse cytokine production directly in vaccine draining lymph nodes (dLN) extracted early after in vivo vaccination. To do this we perform cytokine multiplex analysis of supernatants from whole dLN cell suspensions following a brief ex vivo incubation.
Immunology
Enzymatic Reactions and Detection of C3 Cleavage Fragments
The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an example for this kind of enzymatic reactions.
Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast
G-protein-coupled receptors (GPCRs) are the largest group of cell-surface proteins and are major molecular targets for drug development. The protocol described herein is for the detection of human GPCR signaling in the yeast Saccharomyces cerevisiae. Using Zoanthus sp. green fluorescent protein (ZsGreen) as the reporter, engineered yeast cells expressing human GPCRs emit strong fluorescence in response to stimuli leading to receptor signal activation. This assay method would allow screening for agonistic ligands and critical mutations required for human GPCR signaling.
Microbiology
HIV-1 Fusion Assay
The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).
Endolysin Expression, Purification and Activity Determination by Zymography
Endolysins are peptidoglycan-degrading (muralytic) enzymes produced by many bacteriophages for cell lysis of the host bacterium. The enzymatic activity of muralytic enzymes can be assayed qualitatively using a zymogram containing incorporated peptidoglycan. This protocol describes the expression of a recombinant 6x His-tagged endolysin using an Escherichia coli (E. coli) expression system and native affinity purification of the protein using Ni-NTA agarose. For the zymogram, the protocol details isolation of crude peptidoglycan from the Gram-negative bacterium Rhodobacter capsulatus and the zymography of purified protein and crude cell lysate. Construction of an E. coli BL21 (DE3) pET28-a(+)-derived endolysin-expression system is briefly described.The protocol described here was developed and optimized for the endolysin 555 utilized by the Rhodobacter capsulatus bacteriophage-like gene transfer agent (RcGTA) (Westbye et al., 2013) and to study the muralytic activities of protein P14 of RcGTA (Fogg et al., 2012), but should be transferrable as a general protocol to express and study a variety of endolysins.
VZV Replication Assays
Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the development of disease. This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. As VZV is highly cell-associated in tissue culture, the reporter virus inoculum described here is a preparation of infected cells. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication.
In vitro DNA Polymerization Activity Assay Using Cell-free Extracts
This protocol has been designed to measure the in-vitro DNA polymerization activity in crude cell extracts of the Antarctic bacterium Pseudomonas syrinagae Lz4W. This bacterium can grow at 4 °C with optimum growth rate at 22 °C. The slow growth rate of the bacterium observed at low temperature (4 °C) compared to higher temperature (22 °C) can be attributed to the reduced rate of DNA replication at low temperature. Here we describe a protocol which we have used to quantify the in vitro DNA polymerization of cell extracts at two different temperatures.
Neuroscience
Elevated Plus Maze Test to Assess Anxiety-like Behavior in the Mouse
The elevated plus maze task is a simple method to assess anxiety-like behaviors in rodents. This version describes the procedure used in mice. However, the protocol may also be applied to rats, considering a proportionally larger apparatus (arms: 10 x 50 cm; height: 55 cm). Briefly, the test is performed on a plus-shaped apparatus with two open and two closed arms. The animal is allowed to freely explore the maze for 5 min while the duration and frequency of entries into open and closed arms is recorded. The task is based on an approach-avoidance conflict, meaning that the animal is faced with a struggle between a propensity to explore a novel environment and an unconditioned fear of high and open spaces. Consequently, an anxiety-like state is characterized by increased open arm avoidance, compared to control animals. On account of being a very popular test, there can be considerable variations in the procedures applied across different laboratories. Here we provide a working protocol that has been able to detect both anxiogenic and anxiolyitic drug effects under the specified conditions. Protocol originally published in (Leo et al., 2014).
Plant Science
Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue
Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant tissue. PPO activity can be easily assayed in crude protein extracts from English walnut (Juglans regia) leaves and from many other plant tissue extracts. PPO activity is most commonly measured by spectrophotometric assay, in which the rate of phytomelanin production is quantified, or by oxygen electrode assay, in which the consumption of oxygen by the enzyme is quantified (Figure 1). Though simpler, the utility of the spectrophotometric assay is limited by variation in the absorption maxima of phytomelanins generated from different phenolic substrates. The oxygen electrode assay is generally considered the “gold standard” for measurement of PPO activity, but it is more time consuming and difficult to implement with monophenol substrates, since cresolase activity is typically quite low compared to catecholase activity. This protocol will describe crude protein extraction from walnut leaves, the spectrophotometric assay, and the oxygen electrode assay for determining PPO activity.
Salinity Assay in Arabidopsis
Salinity is an important environmental constraint to crop productivity in arid and semi-arid regions of the world. The evaluation of the responses to salinity of different Arabidopsis ecotypes or transgenic lines is important to identify and investigate the role of different key genes. These new characterized genes involved in the response to salinity stress are of great interest to be incorporated in crops breeding programs. Here we provide a reproducible method to evaluate the performance of Arabidopsis lines to salinity stress by analysing primary and lateral root growth and fresh weight of plants grown under in vitro conditions in growth chambers. Even though NaCl is the most frequent used salinity tests, other salts (e.g. KCl, MgCl2) can be also evaluated by this method. Arabidopsis plants can be maintained for 15-20 days in these conditions, although effects on growth and biomass can be observed, depending on the used salt and concentration, within the first 10 days.
Salinity Assay in Tomato
Tomato is one of the most important horticultural crops worldwide, and is cultivated in semi-arid regions in which soil and groundwater salinity is an increasing limitation to yield. The assessment of the responses of new cultivars to salt and the comparisons among cultivars and wild species are of great interest in tomato breeding. This assay provides a reproducible and reliable method for screening tomato responses to NaCl salinity under hydroponic conditions in growth chambers. Although NaCl is the most commonly used salt in salinity studies, other salts such as Na2SO4, MgCl2 or MgSO4, usually found in saline soils, can also be assayed (Nebauer et al., 2013). Plants can be maintained for 30-45 days under the described conditions, although significant effects on growth can be observed after 10 days, depending on the salt and concentration used.
SGR-based Reporter to Assay Plant Transcription Factor-promoter Interactions
We developed an in vivo method to assay plant transcription factor (TF)–promoter interactions using the transient expression system in Nicotiana benthamiana (N. benthamiana) plants. The system uses the Arabidopsis stay green (SGR) gene as a reporter. Induction of SGR expression in N. benthamiana causes chlorophyll degradation and causes leaves to turn yellow.
Systems Biology
siRNA Screening for Genes Involved in HSV-1 Replication
Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, 2013).