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    A gas trapping method for high-throughput metabolic experiments.
    Biotechniques 2018 Jan 1;64(1):27-29. Epub 2018 Jan 1.
    School of Life and Environmental Sciences, Charles Perkins Centre, The University of Sydney.
    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. Read More

    Standardized cloning vectors for protein production and generation of large gene libraries in Escherichia coli.
    Biotechniques 2018 Jan 1;64(1):24-26. Epub 2018 Jan 1.
    Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany.
    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. Read More

    General and facile purification of dye-labeled oligonucleotides by pH-controlled extraction.
    Biotechniques 2018 Jan 1;64(1):21-23. Epub 2018 Jan 1.
    Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
    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. Read More

    Vital ex vivo tissue labeling and pathology-guided micropunching to characterize cellular heterogeneity in the tissue microenvironment.
    Biotechniques 2018 Jan 1;64(1):13-19. Epub 2018 Jan 1.
    Department of Biomedical Engineering, University of Wisconsin, Madison, WI.
    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. Read More

    Preparing thin cross sections of Arabidopsis roots without embedding.
    Biotechniques 2017 Dec 1;63(6):281-283. Epub 2017 Dec 1.
    Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
    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. Read More

    Processing fixed and stored adipose-derived stem cells for quantitative protein array assays.
    Biotechniques 2017 Dec 1;63(6):275-280. Epub 2017 Dec 1.
    Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI.
    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. Read More

    Linear array of multi-substrate tracts for simultaneous assessment of cell adhesion, migration, and differentiation.
    Biotechniques 2017 Dec 1;63(6):267-274. Epub 2017 Dec 1.
    Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC.
    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. Read More

    Decoding DNA labels by melting curve analysis using real-time PCR.
    Biotechniques 2017 Dec 1;63(6):261-266. Epub 2017 Dec 1.
    Avicor Ltd., Szeged, Hungary.
    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. Read More

    Western blot analysis of cells encapsulated in self-assembling peptide hydrogels.
    Biotechniques 2017 Dec 1;63(6):253-260. Epub 2017 Dec 1.
    School of Materials, The University of Manchester, Manchester, UK.
    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. Read More

    Protocol for adhesion and immunostaining of lymphocytes and other non-adherent cells in culture.
    Biotechniques 2017 Nov 1;63(5):230-233. Epub 2017 Nov 1.
    Division of Dermatology, University of Ottawa, Ottawa, Ontario, Canada.
    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. Read More

    ReViMS: Software tool for estimating the volumes of 3-D multicellular spheroids imaged using a light sheet fluorescence microscope.
    Biotechniques 2017 Nov 1;63(5):227-229. Epub 2017 Nov 1.
    Advanced Research Center on Electronic Systems (ARCES) for Information and Communication Technologies "E. De Castro," University of Bologna, Italy; Department of Computer Science and Engineering (DISI), University of Bologna, Italy.
    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. Read More

    Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing.
    Biotechniques 2017 Nov 1;63(5):221-226. Epub 2017 Nov 1.
    Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY.
    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. Read More

    Mouse Behavior Tracker: An economical method for tracking behavior in home cages.
    Biotechniques 2017 Nov 1;63(5):215-220. Epub 2017 Nov 1.
    Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS.
    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). Read More

    A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research.
    Biotechniques 2017 Nov 1;63(5):205-214. Epub 2017 Nov 1.
    Institute of Virology, University of Ulm, Ulm, Germany.
    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. Read More

    MicroScale Thermophoresis (MST) for studying actin polymerization kinetics.
    Biotechniques 2017 Oct 1;63(4):187-190. Epub 2017 Oct 1.
    Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
    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. Read More

    Genotyping live fish larvae: Non-lethal and noninvasive DNA isolation from 3-5 day old hatchlings.
    Biotechniques 2017 Oct 1;63(4):181-186. Epub 2017 Oct 1.
    Department of Marine Biology, Texas A&M University, Galveston Campus, Galveston, TX; Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX.
    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). Read More

    An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.
    Biotechniques 2017 Oct 1;63(4):174-180. Epub 2017 Oct 1.
    Dokuz Eylul University, School of Medicine, Department of Medical Biochemistry, Izmir, Turkey.
    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. Read More

    Density separation of quiescent yeast using iodixanol.
    Biotechniques 2017 Oct 1;63(4):169-173. Epub 2017 Oct 1.
    Department of Biology.
    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. Read More

    Image-based cell-size estimation for baculovirus quantification.
    Biotechniques 2017 Oct 1;63(4):161-168. Epub 2017 Oct 1.
    University of Tartu, Institute of Chemistry, Tartu, Estonia.
    Measurement of virus concentration is essential for effective virus-based transfection technologies. Here, we describe a user-friendly, image-based cell-size estimation (ICSE) assay for baculovirus quantification that relies on automated determination of cell diameters from bright-field microscopy images. In the ICSE assay, microplate-based imaging systems and our custom ICSE-Tools software enable measurement of cell morphological parameters over time. Read More

    Development and Optimization of a High Titer Recombinant Lentivirus System.
    Biotechniques 2017 Sep 1;63(3):136-138. Epub 2017 Sep 1.
    Mirus Bio LLC, Madison, Wisconsin, USA.
    To enable simple and effective high titer recombinant lentivirus production, we examined key parameters for the generation of lentivirus including: transfection optimization, media change, incubation time and DNA vector selection. These results illustrate the importance of optimizing transfection processes for high titer recombinant lentivirus production. Read More

    Generation of iPS-derived model cells for analyses of hair shaft differentiation.
    Biotechniques 2017 Sep 1;63(3):131-134. Epub 2017 Sep 1.
    Department of Biomedical Chemistry, Graduate School of Science and Technology, Kwansei Gakuin University, 669-1337, Sanda, Japan.
    Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). Read More

    Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.
    Biotechniques 2017 Sep 1;63(3):125-130. Epub 2017 Sep 1.
    Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea.
    Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. Read More

    Direct quantification of protein glycan phosphorylation.
    Biotechniques 2017 Sep 1;63(3):117-123. Epub 2017 Sep 1.
    Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research (CDER), Food and Drug Administration (FDA), Silver Spring, MD.
    Phosphorylation is an important post-translational modification (PTM) of proteins and a critical quality attribute for protein therapeutics, especially if it is required for protein function or sub-cellular targeting. Most current methods to quantify phosphorylation are time-consuming, indirect, or require specific instrumentation and technical skills. Here, we report the adaptation of a phosphate-specific binding dye and common laboratory instruments for quantification of relative amounts of phosphorylated glycans as well as phosphorylation of amino acid residues on the backbones of proteins. Read More

    Construction of a combinatorial library of chimeric tumor-specific promoters.
    Biotechniques 2017 Sep 1;63(3):107-116. Epub 2017 Sep 1.
    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
    Gene therapy is a fast-developing field of molecular medicine. New, effective, and cancer-specific promoters are in high demand by researchers seeking to treat cancer through expression of therapeutic genes. Here, we created a combinatorial library of tumor-specific chimeric promoter modules for identifying new promoters with desired functions. Read More

    [Letter to the Editor] Incorrect assignment of affected nucleotides in footprinting/probing experiments.
    Biotechniques 2017 Sep 1;63(3):105-106. Epub 2017 Sep 1.
    Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
    Address correspondence to Sergey Belikov or Lars Wieslander, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: sergey.belikov@su. Read More

    Overcoming qRT-PCR interference by select carbon nanotubes in assessments of gene expression.
    Biotechniques 2017 Aug 1;63(2):81-84. Epub 2017 Aug 1.
    Department of Environmental and Global Health, University of Florida.
    Nanomaterials (NMs) of various types, including carbon nanotubes (CNTs), can interfere with standard quantitative real-time PCR (qRT-PCR) assays, resulting in inaccurate gene expression measurements; however, the precise step in the qRT-PCR pipeline where this interference occurs has not been well described. Here, we investigated where in the process surface-oxidized multi-walled CNTs (oxMWNTs) inhibited qRT-PCR measurement of the expression of the housekeeping gene GAPDH and explored several strategies to minimize such inhibition. We determined that the interference occurred during the reverse transcription (RT) step and found that doubling reaction reagents or adding BSA successfully mitigated the inhibition. Read More

    BODIPY-based dye for no-wash live-cell staining and imaging.
    Biotechniques 2017 Aug 1;63(2):77-80. Epub 2017 Aug 1.
    M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation.
    In nonpolar solvents, hydrophobic organic fluorophores often show bright fluorescence, whereas in polar media, they usually suffer from aggregation-caused quenching (ACQ). Here, we harnessed this solvatochromic behavior of a 1,3,5,7-tetramethyl-BODIPY derivative for cell staining and applied it to live-cell imaging and flow cytometry. As opposed to commercially available dyes, this BODIPY derivative showed excellent contrast immediately after staining and did not require any wash-off. Read More

    Spin infection enables efficient gene delivery to muscle stem cells.
    Biotechniques 2017 Aug 1;63(2):72-76. Epub 2017 Aug 1.
    Stem Cell Institute.
    Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. Read More

    Ion-exchange chromatography purification of extracellular vesicles.
    Biotechniques 2017 Aug 1;63(2):65-71. Epub 2017 Aug 1.
    Institute for the Application of Nuclear Energy, INEP, University of Belgrade, Belgrade, Serbia.
    Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. Read More

    Preparation of highly multiplexed small RNA sequencing libraries.
    Biotechniques 2017 Aug 1;63(2):57-64. Epub 2017 Aug 1.
    Lund University Cancer Center, Faculty of Medicine, Department of Clinical Sciences Lund, Oncology and Pathology, Lund, Sweden.
    MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Read More

    Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts.
    Biotechniques 2017 Jul 1;63(1):37-39. Epub 2017 Jul 1.
    Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
    Commercially available lipid-based transfection reagents are widely used to deliver DNA to cells. However, these lipid-based transfection reagents show poor gene transfer efficiency in primary cells. Here, we demonstrate a simple method to improve gene transfer efficiency in primary fibroblasts and hepatoblasts using a combination of lipid-based transfection reagents. Read More

    Counting nuclei released from microcarrier-based cultures using pro-fluorescent nucleic acid stains and volumetric flow cytometry.
    Biotechniques 2017 Jul 1;63(1):34-36. Epub 2017 Jul 1.
    Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
    Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/automated imaging system or a Coulter counter, are imprecise, laborious and, limited in throughput. Here, we describe the use of high-affinity pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcarriers with improved precision and high sample throughput. Read More

    An in vitro technique to identify the RNA binding-site sequences for RNA-binding proteins.
    Biotechniques 2017 Jul 1;63(1):28-33. Epub 2017 Jul 1.
    Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
    RNA-protein interactions play a major role in gene regulation. Although many techniques to analyze RNA-protein interactions have been developed, noteworthy challenges such as determining the RNA sequences that bind RNA-binding proteins (RBPs) remain unsolved. Here, we describe a novel technique using a 4-thio-uridine-incorporated RNA pool to identify the RBP-binding consensus sequences for RBPs produced by in vitro transcription and translation. Read More

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