Methods in molecular biology (Clifton, N.J.) (Meth Mol Biol)

Publisher: Humana Press

Current impact factor: 1.29

Impact Factor Rankings

Additional details

5-year impact 0.00
Cited half-life 0.00
Immediacy index 0.00
Eigenfactor 0.00
Article influence 0.00
Other titles Methods in molecular biology (Clifton, N.J.), Methods in molecular biology
ISSN 1940-6029
OCLC 24839341
Material type Series
Document type Journal / Magazine / Newspaper

Publisher details

Humana Press

  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
  • Conditions
    • Authors own final version only can be archived
    • Publisher's version/PDF cannot be used
    • On author's personal website immediately
    • On any open access repository after 12 months from publication
    • Published source must be acknowledged
    • Must link to publisher version
    • Set phrase to accompany link to published version: The original publication is available at www.springerlink.com
    • Articles in some journals can be made Open Access on payment of additional charge
    • 'Humana Press' is an imprint of 'Springer Verlag (Germany)'
  • Classification
    green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.
    No preview · Article · Feb 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous(®)-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Proteolysis has been cited as an important contributor to cancer initiation and progression. One can take advantage of tumor-associated proteases to selectively deliver imaging agents. Protease-activated imaging systems have been developed using substrates designed for hydrolysis by members of the matrix metalloproteinase (MMP) family. We presently describe approaches by which one can optically image matrix metalloproteinase activity implicated in breast cancer progression, with consideration of selective versus broad protease probes.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Matrix metalloproteinases are endopeptidases responsible for remodeling of the extracellular matrix and have been identified as critical contributors to breast cancer progression. Gelatin zymography is a valuable tool which allows the analysis of MMP expression. In this approach, enzymes are resolved electrophoretically on a sodium dodecyl sulfate-polyacrylamide gel copolymerized with the substrate for the MMP of interest. Post electrophoresis, the enzymes are refolded in order for proteolysis of the incorporated substrate to occur. This assay yields valuable information about MMP isoforms or changes in activation and can be used to analyze the role of MMPs in normal versus pathological conditions.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNA (miRNA) analysis has evolved over the past two decades to become a highly specialized field with broad-reaching applications across a multitude of diseases and cellular processes. The choice of an applicable approach for miRNA quantification will depend on a variety of factors such as cost, time constraints, and throughput. Here, we describe the methods of total RNA isolation, AGO2-bound RNA isolation, miRNA polyadenylation, miRNA-cDNA synthesis, and quantitative real-time polymerase chain reaction for the detection of known miRNAs in cultured cells or xenograft tissues of breast cancer.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Current standard of care requires that HER2 gene testing be performed on all newly diagnosed invasive breast cancers in order to determine eligibility for anti-HER2 antibody therapy and should be performed in accordance with current ASCO-CAP guidelines (Hammond et al., J Clin Oncol 29(15):e458, 2011; Wolff et al., J Clin Oncol 31(31):3997-4013, 2013). Here we describe a HER2 FISH methodology to evaluate HER2 gene status in FFPE breast tumor specimens.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumor microenvironment composition and architecture are known as a major factor in orchestrating the tumor growth and its response to various therapies. In this context, in vivo studies are necessary to evaluate the responses. However, while tumor cells can be of human origin, tumor microenvironment in the in vivo models is host-based. On the other hand, in vitro studies in a flat monoculture of tumor cells (the most frequently used in vitro tumor model) are unable to recapitulate the complexity of tumor microenvironment. Three-dimensional (3D) in vitro cell cultures of tumor cells have been proven to be an important experimental tool in understanding mechanisms of tumor growth, response to therapeutics, and transport of nutrients/drugs. We have recently described a novel tool to create 3D co-cultures of tumor cells and cells in the tumor microenvironment. Our method utilizes magnetic manipulation/levitation of the specific ratios of tumor cells and cells in the tumor microenvironment (from human or animal origin) aiding in the formation of tumor spheres with defined cellular composition and density, as quickly as within 24 h. This chapter describes the experimental protocols developed to model the 3D structure of the cancer environment using the above method.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cell migration is a critical determinant of cancer invasion and metastasis. Drugs targeting cancer cell migration have been hindered due to the lack of effective assays for monitoring cancer cell migration. Here we describe a novel method to microscopically monitor cell migration in a quantitative fashion. This assay can be used to study genes involved in cancer cell migration, as well as screening anticancer drugs that target this cellular process.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: The in situ observation on the tissues, such as histopathology, immunohistochemistry (IHC), immunofluorescence (IF), and in situ hybridization (ISH), is one of the most important methods in the biomedical scientific research. In this chapter we introduce the most often used methods-hematoxylin and eosin (H&E) and double IF staining. H&E staining is used for general morphology by which the different pathological types of breast lesions are identified. The double IF staining is often used to study the protein-protein interaction on tissues for signaling mechanisms. This chapter also includes the histopathology of primary or simplified breast lesion types that is essential for applying the above methods and the reclassification of breast cancers by molecular markers.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Unraveling DNA methylation profile of tumor is important for the diagnosis and treatment of cancer patients. Because of the heterogeneity of clinical samples, it is very difficult to get methylation profile of only tumor cells. Laser capture Microdissection (LCM) is giving us a chance to isolate the DNA only from the tumor cells without any stroma cell's DNA contamination. Once we capture the breast tumor cells, we can isolate the genomic DNA which is followed by the bisulfite treatment in which unmethylated cytosines of the CG pairs are converted into uracil; however, methylated cytosine does not go into any chemical change during this reaction. Next, bisulfite treated DNA is used in the regular PCR reaction to get a single band PCR amplicon which will be used as a template for the pyrosequencing. Pyrosequencing is a powerful method to make a quantitative methylation analysis for each specific CG pair.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Metastasis is the major cause of breast cancer deaths. To spread from the primary tumor sites to distant tissues, solid tumor cells need to degrade the surrounding extracellular matrix (ECM). The protrusive membrane structures named invadopodia have been shown to play a critical role in the degradation of the ECM and invasion of invasive cancer cells. In this chapter, we describe a detailed protocol to examine invadopodia in human breast cancer cells.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Models of tumor angiogenesis have played a critical role in understanding the mechanisms involved in the recruitment of vasculature to the tumor mass, and have also provided a platform for testing antiangiogenic potential of new therapeutics that combat the development of malignant growth. In this regard, the chorioallantoic membrane (CAM) of the developing chick embryo has proven to be an elegant model for investigation of angiogenic processes. Here, we describe methods for effectively utilizing the preestablished vascular network of the chick CAM to investigate and quantify tumor-associated angiogenesis in a breast tumor model.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Numerous technological improvements, including progress in vector design, simplification of virus isolation techniques, and advancements in molecular biology and cell culture technologies, have greatly facilitated the use of the baculovirus-insect cell system for routine production of recombinant proteins. This chapter outlines the basic techniques for small-scale protein production using the Baculovirus Expression Vector System (BEVS), including protocols for titer estimation in 96-well plates, expression optimization in 24-well plates, and recombinant protein expression from adherent and suspension cultures in six-well plates and in 50 mL insect cell cultures.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Probing the baculovirus infection process is essential in optimizing recombinant protein production. Typically, researchers monitor the infection process in stirred reactors that contain cells that have been infected at different times after virus inoculation, particularly if cells pass the primary infection and become infected by progeny virus. This chapter describes several alternative bioreactor systems for baculovirus infection. We provide an example alternative system that holds promise to avoid asynchronous distributions in infection time. Namely, we describe a two-stage reactor system consisting of an upstream continuous stirred tank reactor and a downstream tubular reactor with segmented plug flow for probing baculovirus infection and production.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Immunohistochemical analysis has been a key clinical tool that shows the protein expression of molecular markers. Expression of molecular markers in breast pathology has been used to distinguish breast cancers from benign lesions, classify subtypes of breast cancers, and determine therapeutic intervention. It is a relatively fast and efficient option in stratifying breast lesions to assist in both determining pathology diagnosis and offer strategies to the best course of clinical action. In this chapter, we discuss the use of immunohistochemistry testing for some of the key molecular markers involved in breast pathology that are crucial for classifying breast cancers and the guidelines for the interpretation of testing results that assist in clinical management.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Expression of recombinant proteins in insect cells infected with baculoviruses is commonplace. This system provides an easy way to generate a significant amount of properly folded, functional protein with proper posttranslational modifications and can be used to effectively produce multi-protein complexes. This chapter describes an alternative method of expressing high order protein complexes in insect cell culture. Specific examples involving the expression of 5- and 8-protein complexes are discussed.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nanoparticle delivery is becoming an increasingly more valuable technique in cancer drug treatments. The use of fluorescent probes, in particular, can provide noninvasive strategies to interrogate the internalization mechanisms of cancer cells and aid in drug design. Here we describe the delivery of fluorescently labeled polysaccharide-based nanoparticles to breast cancer cells in vitro and their subsequent immunofluorescence microscopy examination. The description of the synthesis, preparation, and delivery of the nanoparticles can be widely applicable to other in vitro drug delivery studies.
    No preview · Article · Jan 2016 · Methods in molecular biology (Clifton, N.J.)