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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
(no author information available yet)
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.
October 1, 2017: BioTechniques
Andrea Topf, Peter Franz, Georgios Tsiavaliaris
Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation...
October 1, 2017: BioTechniques
G Janelle Espinoza, J Michael Poland, Jaime R Alvarado Bremer
Genotyping fish larvae is a valuable technique for numerous fields of study. While methods for collecting DNA from early stage larvae have been published, a non-lethal, non-invasive genotyping protocol for hatchlings that is amenable to high-throughput approaches is desirable. Here, we describe a method to individually genotype live, free-swimming, early fish larvae by characterizing their environmental DNA (eDNA). We demonstrate the utility of the method by assigning parentage to a sample (n = 50) of 3-5-day-old sheepshead minnow (Cyprinodon variegatus) larvae hatchlings, with very high rates of genotyping success (98%) and survival (92%) using mitochondrial and microsatellite DNA data...
October 1, 2017: BioTechniques
Seniz Inanc, Didem Keles, Gulgun Oktay
Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13...
October 1, 2017: BioTechniques
Ishtiaque Quasem, Christopher J Luby, Charles R Mace, Stephen M Fuchs
As yeast are starved of nutrients, they enter G0, a quiescent state. Quiescent yeast (Q) cells retain viability for extended periods of time and resume growth following supplementation of missing nutrients. As such, Q cells have become a valuable model for studying longevity and self-renewal of chronologically aged cells. Traditional isolation of Q cells involves a relatively long centrifugation time through a continuous density gradient. Here, we describe a rapid and cost-effective Q-cell isolation technique that uses a single-density, one-step gradient prepared from media containing iodixanol...
October 1, 2017: BioTechniques
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