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James R Krycer, Ciana Diskin, Marin E Nelson, Xiao-Yi Zeng, Daniel J Fazakerley, David E James
Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. We used this trap design to measure CO2 production from glucose and fatty acid metabolism, as well as hydrogen sulfide production from cysteine-treated cells...
January 1, 2018: BioTechniques
Bettina Rohweder, Florian Semmelmann, Christiane Endres, Reinhard Sterner
Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E...
January 1, 2018: BioTechniques
So Young Bak, Jihee Hwang, Sohyeon Bae, Soonkyu Lim, Younggyu Kim, Khalid Alzahrani, Rizwan Wahab, Abdulaziz Alkhedhairy, Seong Keun Kim
Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. When the pH of an aqueous solution containing dye-labeled DNA and unreacted free dye is lowered, and the solution is mixed with a hydrophobic organic solvent (butanol), the neutral free dye is preferentially dissolved in the organic phase, leaving behind the hydrophilic dye-labeled DNA in the aqueous phase...
January 1, 2018: BioTechniques
Brian P Johnson, Ross A Vitek, Peter G Geiger, Wei Huang, David F Jarrard, Joshua M Lang, David J Beebe
Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution; however, this heterogeneity it is difficult to characterize. Here, we present a new approach-pathology-guided micropunching (PGM)-that enables identification and characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of live-cell tissue labeling using fluorescent antibodies/small molecules to identify heterogeneous foci (e...
January 1, 2018: BioTechniques
Hannah Martin Lawrenz
Newly engineered bacterial strains propel an immune-mediated cancer therapy toward clinical applications. Hannah Martin Lawrenz reports on the latest developments.
January 1, 2018: BioTechniques
Kristie Nybo
A 200-year-old observation might provide a new way to eliminate tumors by infecting cancer patients with bacteria. Kristie Nybo explores a new approach that could transform cancer treatment.
January 1, 2018: BioTechniques
Naoyuki Sotta, Toru Fujiwara
Here, we describe a method for obtaining thin cross sections of Arabidopsis thaliana roots without fixation and embedding. Roots were grown in pinholes made in a solidified growth medium, and cross sections were prepared without pretreatment. Using this method, we detected unique distributions of two polar-localized proteins-green fluorescent protein (GFP)-tagged BOR1 and NIP5;1-with less sample preparation time than conventional methods. This method is simple, rapid, and yields high-quality cross-section images that are free from artifacts commonly associated with embedding or the sample preparation procedures used in many conventional methods...
December 1, 2017: BioTechniques
Jessica S Sadick, Eric M Darling
Accurately characterizing cellular subpopulations is essential for elucidating the mechanisms underlying normal and pathological biology. Isolation of specific cell types can be accomplished by labeling unique cell-associated proteins with fluorescent antibodies. Cell fixation is commonly used to prepare these samples and allow for long-term storage, but this poses challenges for subsequent protein analysis. We previously established the FITSAR (formaldehyde-fixed intracellular target-sorted antigen retrieval) method, in which protein can be isolated and characterized from fixed, enriched cell subpopulations...
December 1, 2017: BioTechniques
Ricardo A Moreno-Rodriguez, Edward L Krug, Leticia Reyes, Roger R Markwald
Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Here, we present a method for assessing cell adhesion, migration, and differentiation in up to 20 different test conditions simultaneously, using only minute amounts of target substrate...
December 1, 2017: BioTechniques
József A Balog, Liliána Z Fehér, László G Puskás
Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2...
December 1, 2017: BioTechniques
Kyle A Burgess, Aline F Miller, Delvac Oceandy, Alberto Saiani
Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct...
December 1, 2017: BioTechniques
Sarah Webb
Directed evolution is poised to change small molecule discovery and provide greater access to "drug space." Sarah Webb looks into the evolving drug discovery landscape.
December 1, 2017: BioTechniques
Anna Hiltunen, Malena Skogman, Pia M Vuorela, Adyary Fallarero
No abstract text is available yet for this article.
November 1, 2017: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
November 1, 2017: BioTechniques
Matthew Tsang, Jennifer Gantchev, Feras M Ghazawi, Ivan V Litvinov
Immunostaining of non-adherent cells is commonly performed after adhesion of cells onto microscope slides either using cytocentrifugation or with the help of charged coating substrates. These techniques, however, require either specialized equipment or significant preparation time. Here, we describe a method for immunofluorescent staining of lymphocytes within multi-well culture plates, where cells suspended in phosphate buffered saline (PBS) are adhered to either the plastic well bottom or glass coverslips by gravity sedimentation...
November 1, 2017: BioTechniques
Filippo Piccinini, Anna Tesei, Michele Zanoni, Alessandro Bevilacqua
Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. However, there remains a lack of validated methods for quantifying the volumes of 3-D multicellular aggregates...
November 1, 2017: BioTechniques
Jungeui Hong, David Gresham
Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Misidentification of these molecules as PCR duplicates can introduce unforeseen biases during analyses. Here, we developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed, single-index sequencing...
November 1, 2017: BioTechniques
Sudheer K Tungtur, Natsuko Nishimune, Jeff Radel, Hiroshi Nishimune
Analysis of mouse behavior often requires expensive equipment and transfer of the mice to new test environments, which could trigger confounding behavior alterations. Here, we describe a system for tracking mouse behavior in home cages using a low-cost USB webcam and free software (Fiji and wrMTrck). We demonstrate the effectiveness of this method by tracking differences in distance traveled, speed, and movement tracks between wild-type mice and amyotrophic lateral sclerosis (ALS) model mice (SOD1G93A).
November 1, 2017: BioTechniques
Kerstin Laib Sampaio, Anja Weyell, Narmadha Subramanian, Zeguang Wu, Christian Sinzger
For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2-US6 gene region (yielding TB40-BACKL7), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2-US6 region was restored, resulting in BAC clone TB40-BACKL7-SE When this BAC clone was transfected into fibroblasts at efficiencies >0...
November 1, 2017: BioTechniques
Nathan Blow
Advances in fluorescent dye and protein design have led to a wider color palette for microscopy. Nathan Blow explores how this happened and what is still to come.
November 1, 2017: BioTechniques
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