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Past Issue in 2015
Volume: 5, Issue: 22
Cancer Biology
In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer
The in vitro limiting dilution assay is used to determine the colorectal cancer initiating cell (CC-IC) frequency of a CC-IC enriched suspension culture, grown in growth factor enriched serum free media. The in vivo limiting dilution assay is used to determine the colorectal cancer initiating cell frequency of a primary colorectal cancer sample or an established suspension cell line using immunocompromised murine xenograft models. In vitro and in vivo limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or genetic knockdown strategy on the initiating cell frequency of a population of CC-ICs or colorectal cancer sample, respectively.
Telomere Restriction Fragment (TRF) Analysis
While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells.Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.
Telomerase Repeated Amplification Protocol (TRAP)
Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al., 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase- consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC- counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al., 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non-telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is analyzed by electrophoresis. TSNT is, an internal standard control, amplified by TS primer. NT is its own reverse primer, which is not a substrate for telomerase. These primers are used to identify false-negative results by if the gel lacks internal control bands.
13C Tracer Studies of Metabolism in Mouse Tumor Xenografts
Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including 13C6-glucose and 13C5,15N2-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors.
Telomere Dysfunction Induced Foci (TIF) Analysis
Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells.Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.
Immunology
Skin Wound Healing Model - Excisional Wounding and Assessment of Lesion Area
This protocol focus on the most common surgical mouse model of cutaneous excisional wound healing used to study the cellular and molecular pathways involved in wound repair and regeneration as well as in translational applications such as the evaluation of new therapeutic modalities. This model allows the monitoring of the wound closure and the tissue collection for histological and molecular analyses. Briefly, full skin thickness excisional wounds are created on the dorsum of the mouse as the excision extends through the panniculosus carnosus. Wounds larger and minor diameters are then regularly measured and wound closure rate is calculated based on wound area relative to the original size.
Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities
The inflammatory response is essential to the reestablishment of cutaneous homeostasis following injury. In this context, leukocytes arrive at the wound site and orchestrate essential events in the wound healing process. Therefore, the quantification of specific subsets of inflammatory cells in the wound tissue is of considerable interest. The current protocol focus on a quantitative index of neutrophils and macrophages accumulation within skin lesions by measuring the specific activity of the marker enzymes Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG), respectively. MPO is present in high levels in the azurophilic granules of neutrophils and NAG in lysosomes of activated macrophages. These methods allow the indirect estimation of the abundance of neutrophils and macrophages accumulated into the skin.
Permanent Occlusion of the Left Anterior Coronary Artery in the Rat
Left ventricular (LV) remodeling occurs in many patients after myocardial infarction (MI). LV remodeling is characterized by progressive ventricular dilatation and contractile dysfunction, consequently to cardiomyocyte hypertrophy and fibrosis. Despite reperfusion therapies, this pathophysiological process is the main cause of cardiac evolution toward heart failure. Moreover, the outcome of patients after MI is largely dependent on the initial cardiac injury. Thus, this is of major clinical interest to develop new pharmacological strategies to limit infarct size and prevent or reverse left ventricular remodeling. Such preclinical cardiovascular treatments are often tested in rodents. The rat model of myocardial infarction is commonly used. In this model, the permanent ligation of the left anterior descending coronary artery is performed (Bousquenaud et al., 2013a). After being used to this surgical technique and experimented, the operator will need 20 min per rat from the anesthesia to the rat recovering.
Microbiology
Purification of Bacterial RNA from Infected Macrophages
Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).
An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast
Heterologous expression of genes in budding yeast Saccharomyces cerevisiae (S. cerevisiae) is especially suitable to functionally study the corresponding encoded protein at the cellular level (Bonneaud et al., 1991). This is mainly because many strains defective in specific activities are available and could be complemented by homologous genes existing across the eukaryotic kingdom (http://www.yeastgenome.org/). However, the protocol we describe here is not a complementation but a “gain-of-function” assay. It is based on a drop-test assay that we have set up to assess the cellular zinc tolerance conferred by the expression of heterologous genes in the wild-type S. cerevisiae. Different dilutions of a yeast culture expressing the heterologous gene of interest are grown on a range of zinc-enriched plates, and are then compared to the control yeast expressing the empty vector. Working with different concentrations of both yeast and zinc are essential to succeed in describing zinc tolerance phenotype upon yeast transformation (Mirouze et al., 2006). This test has also proven to be valuable to differentiate among related members of gene families as exemplified for Arabidopsis Plant Defensin type1 (Shahzad et al., 2013).
Neuroscience
Locomotor Coordination Assay in Rats
Neonatal hypoxia-ischemia (HI) affects 60% of low birth weight infants and up to 40% of preterm births. Cell death and brain injury after HI have been shown to cause long-lasting neurological deficits. Two motor coordination tests on rats that had been exposed to HI on postnatal day 7 (P7) showed that HI in the P7 rat is associated with significant motor coordination impairment. These results call attention to the risks associated with perinatal ischemia and the need for proper treatment to reverse HI-induced deleterious effects.
Plant Science
Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS
One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.
Arabidopsis Leaf Explant Culture
In this protocol, Arabidopsis leaf explant culture is described using an adaptation of a previous method (Hu et al., 2000). Cells from the cut edges of leaf explant are able to proliferate and subsequently form calli on the callus induction medium, in which is supplemented with 2,4-D and 6-benzyl aminopurine [6-BA]. 2,4-D, one of the artificial auxin, is able to promote cell mitosis at low concentration. 6-BA, the first generation of synthetic cytokinin, plays an important role in plant cell division. 2,4-D in combination with 6-BA can effectively induce callus formation (Rashmi and Trivedi, 2014). The aim of this protocol is to analyze cell division competence of Arabidopsis plants with different genotypes. This protocol can be modified and applied to culture explants from other types of plant tissues, such as root and stem.
Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase
Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance. In this protocol, we describe the expression, purification and activity assay of the heterologously expressed GTPase RabA4c from A. thaliana based on the publication Ellinger et al. (2014). We fused RabA4c to the fluorophore mCitrine and expressed this protein in the yeast strain Pichia pastoris GS115. For purification of RabA4c, we used the GFP-Trap_A kit (Chromo Tek) which specifically binds to GFP derivatives like mCitrine. The enzyme activity assay was done by using the GTPase Assay Kit from Innova Biosciences. In general, we followed the instructions made by the manufacturers.
Quantifying the Permeability of the Apoplastic Water Barrier in Cosmos Petals
The capacity of plants to minimize uncontrolled water loss is essential for survival in adverse and changing climatic conditions. In order to assess and compare the effectiveness of apoplastic barriers to water, the permeability of the barrier must first be quantified. Studies have accomplished this directly by quantifying tritium flux or indirectly by measuring the influx/efflux of water surrogates such as dyes, chlorophyll, and herbicides. Other studies have relied on comparative methods such as survival rates after drought. These methods rely on radioactive material, correlations, or qualitative comparisons. However, a quantitative method is necessary that directly measures water efflux and that allows easy comparisons within and between experiments, plant parts, plant species, and especially research laboratories. Here we outline in detail a gravimetric protocol first described by Schönherr and Lendzian (1981) that can be set up in less than half a day and completed in one to ten days depending on the plant barrier. This approach has been used in numerous studies on leaf and fruit cuticles and recently also on petals from cosmos (Cosmos bipinnatus; Buschhaus et al., 2015).