Springer protocols feed by Cancer Research 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Detection and Quantification of Apoptosis in Primary Cells Using Taqman® Protein Assay
There are several methods to detect apoptosis using cleaved caspase-3 and each harbors its own advantages and disadvantages. When primary cell cultures are used, the disadvantages of the standard methods can make apoptosis detection difficult due to their slow growth rate and replicative senescence, thereby limiting the available cell number and experiment time span. In this chapter, we describe apoptosis detection and quantification using an innovative method named TaqMan® protein assay. TaqMan® protein assay uses antibodies and proximity ligation for quantitative real-time PCR. Biotinylated antibodies are labeled...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Detection of End-Stage Apoptosis by ApopTag® TUNEL Technique
DNA fragmentation, the end stage of apoptosis, is the measure of ultimate demise of the cell. A convenient method for examining apoptosis via DNA fragmentation is by the Terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay where the DNA strand breaks are detected by enzymatically labeling the free 3′-OH termini with modified nucleotides. ApopTag® kits detect single-stranded and double-stranded breaks associated with apoptosis. This technique is also helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
A Multiplexed Method for Kinetic Measurements of Apoptosis and Proliferation Using Live-Content Imaging
We describe a kinetic multiplex assay incorporating the CellPlayerTM NucLight Red reagent to measure proliferation and the CellPlayerTM Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyteTM ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
“Multiplexed Viability, Cytotoxicity, and Caspase Activity Assays”
Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice is not only more cost efficient, but also provides more information about a compound or treatment. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization, which may help correct for differences in cell number. (Source: Springer protoc...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Flow Cytometry Enumeration of Apoptotic Cancer Cells by Apoptotic Rate
Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Caspase-3 Activation Is a Critical Determinant of Genotoxic Stress-Induced Apoptosis
Apoptosis can be measured by number of methods by taking advantage of the morphological, biochemical, and molecular changes undergoing in a cell during this process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the f...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
PET Imaging for Tyrosine Kinase Inhibitor (TKI) Biodistribution in Mice
Receptor tyrosine kinases play a critical role in cell growth, survival, and proliferation, and are considered potential molecular targets for the treatment of cancer. Although several tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, have demonstrated clinical efficacy via the inhibition of the epidermal growth factor receptor (EGFR), most TKIs are only effective in a small proportion of patients. Positron emission tomography (PET) imaging is a methodology of molecular imaging based on nuclear imaging. PET imaging in combination with radiolabeled TKIs improves accuracy of quantitative imaging strategies ...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Measuring Cardiac Autophagic Flux In Vitro and In Vivo
Autophagy is a lysosomal-dependent catabolic pathway that recycles various cytoplasmic-borne components, such as organelles and proteins, through the lysosomes. This process creates energy and biomolecules that are used to maintain homeostasis and to serve as an energy source under conditions of acute stress. Autophagic flux is a measure of efficiency or throughput of the pathway. Here, we describe a method for determining autophagic flux in vitro and in vivo using the autophagosomal/lysosomal fusion inhibitors chloroquine or bafilomycin A1 and then probing for the autophagosomal marker LC3-II via Western Blot. (Source: Sp...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Optical Imaging of Ovarian Cancer Using HER-2 Affibody Conjugated Nanoparticles
Computed Tomography (CT), Ultrasound (US), and Magnetic Resonance Imaging (MRI) have been the mainstay of clinical imaging regimens for the detection of ovarian cancer. However, without tumor specific contrast enhancement, these imaging modalities lack specificity and sensitivity in the detection of small primary and disseminated tumors in the peritoneal cavity. Herein, we illustrate a fairly new near infrared (NIR) optical imaging approach developed in our laboratory for the noninvasive detection of ovarian tumors using a HER-2 targeted nanoparticle-based imaging agent in an orthotopic mouse model of ovarian cancer. We us...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Analysis of Autophagosome Formation Using Lentiviral Biosensors for Live Fluorescent Cellular Imaging
In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Using the Peggy Simple Western System for Fine Needle Aspirate Analysis
Simple Western™ assays are capillary-based electrophoretic immunoassays, similar in scope to SDS-PAGE (molecular weight separation, “size”) and IEF (isoelectric focusing, “charge”) immunoblotting. The enhanced sensitivity and automation of the Simple Western makes it better suited to cancer diagnostics and research than the traditional Western platform. Because of its smaller sample volume requirements, primary cells, such as those obtained from fine needle aspirates (FNAs), and solid tumor slices may be used to generate quantitative comparable data. The Peggy™ instrument is capable of p...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Laser Capture Microdissection for Gene Expression Analysis
Laser capture microdissection (LCM) is an excellent and perhaps the only platform to isolate homogeneous cell populations from specific microscopic regions of heterogeneous tissue section, under direct microscopic visualization. The basic operations of the LCM system are based on (a) microscopic visualization of phenotypically identified cells of interest, (b) selective adherence of cells to a melting thermolabile film/membrane using a low-energy infrared laser (IR system) or photovolatization of cells within a selected region (UV system), (c) capturing or catapulting of structurally intact cells from a stained tissue sect...
Source: Springer protocols feed by Cancer Research - October 15, 2014 Category: Cancer & Oncology Source Type: news
Synthesis of Stabilized Alpha-Helical Peptides
Stabilized alpha-helical (SAH) peptides are valuable laboratory tools to explore important protein–protein interactions. Whereas most peptides lose their secondary structure when isolated from the host protein, stapled peptides incorporate an all-hydrocarbon “staple” that reinforces their natural alpha-helical structure. Thus, stapled peptides retain their functional ability to bind their native protein targets and serve multiple experimental uses. First, they are useful for structural studies such as NMR or crystal structures that map and better define binding sites. Second, they can be used to identify ...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Targeted Genome Modification via Triple Helix Formation
Triplex-forming oligonucleotides (TFOs) are capable of coordinating genome modification in a targeted, site-specific manner, causing mutagenesis or even coordinating homologous recombination events. Here, we describe the use of TFOs such as peptide nucleic acids for targeted genome modification. We discuss this method and its applications and describe protocols for TFO design, delivery, and evaluation of activity in vitro and in vivo. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Cancer Metabolism: Cross Talk Between Signaling and O-GlcNAcylation
Cancer cells exhibit a unique metabolic shift to aerobic glycolysis that has been exploited diagnostically and therapeutically in the clinic. Oncogenes and tumor suppressors alter signaling pathways that lead to alterations of glycolytic flux. Stemming from glycolysis, the hexosamine biosynthetic pathway leads to elevated posttranslational addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins, many of which regulate signaling pathways. This unit outlines techniques used to detect metabolic alterations in cancer cells, regulation by signaling pathways, and cellu...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
