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L Noriega-Calixto L, María E Campos, Gladys I Cassab
The system for analyzing the hydrotropic curvature with a moisture gradient in wild-type Arabidopsis roots was modified. Optimal conditions were determined for detecting a hydrotropic curvature of 90° just after 4 h of stimulation. This system only requires 15 ml of a solution of K2 CO3 with a density of 1.48 g·ml-1 to generate a rapid moisture gradient inside a square Petri dish without decreasing root growth. In this, the root growth rate observed in hydrostimulated wild-type and miz1 mutant, utilized as a negative control, increases sixfold compared with those roots examined using the former method...
January 11, 2019: BioTechniques
Yuko Takagi, Hirokazu Nosato, Motomichi Doi, Koji Furukawa, Hidenori Sakanashi
Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays...
December 13, 2018: BioTechniques
J A Sargent, Vhj Roberts, J E Gaffney, A E Frias
Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy...
October 29, 2018: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
January 2019: BioTechniques
Michael J Lowden, Kevin A Henry
Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies' MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases)...
December 2018: BioTechniques
Simran J Kaur, Vladimir Gilman, Minh Duong, David M Asher, Luisa Gregori
Staphylococcus epidermidis is the most common transfusion-associated pathogen contaminating platelet concentrates. Methods to reduce or eliminate contaminating bacteria from platelet units are critical for improving the safety of blood transfusions. We used rapid isolation of DNA aptamers (RIDA) to identify single-stranded (ss)DNA aptamers as ligands that specifically bind to S. epidermidis. Five target-specific ssDNA aptamers (76 mer) were obtained under stringent selection conditions. Aptamer SE43 demonstrated higher binding affinity compared with scrambled control...
December 2018: BioTechniques
Clinton L Cario, John S Witte
Sample automation and management is increasingly important as the number and size of population-scale and high-throughput projects grow. This is particularly the case in large-scale population studies where sample size is far outpacing the commonly used 96-well plate format. To facilitate management and transfer of samples in this format, we present Samasy, a web-based application for the construction of a sample database, intuitive display of sample and batch information, and facilitation of automated sample transfer or subset...
December 2018: BioTechniques
Cara Francesca Smith, Mitchell E McGlaughlin, Stephen P Mackessy
DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only...
December 2018: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
December 2018: BioTechniques
Kai Hung Tiong, Jit Kang Chang, Dharini Pathmanathan, Muhammad Zaki Hidayatullah Fadlullah, Pei San Yee, Chee Sun Liew, Zainal Ariff Abdul Rahman, Kheng Ling Beh, Sok Ching Cheong
We describe a novel automated cell detection and counting software, QuickCount® (QC), designed for rapid quantification of cells. The Bland-Altman plot and intraclass correlation coefficient (ICC) analyses demonstrated strong agreement between cell counts from QC to manual counts (mean and SD: -3.3 ± 4.5; ICC = 0.95). QC has higher recall in comparison to ImageJauto , CellProfiler and CellC and the precision of QC, ImageJauto , CellProfiler and CellC are high and comparable. QC can precisely delineate and count single cells from images of different cell densities with precision and recall above 0...
December 2018: BioTechniques
Freya Leask
Freya Leask explores developments in cell culture and stem cell research that are revolutionizing how diseases are studied and treated.
December 2018: BioTechniques
Matthew Pittman, Yun Chen
In this study, economic magnetic tweezers (EMT) with a sharp gradient field were designed and built, in order to facilitate accurate force measurement. Our design costs less than 40 USD and is easy to mount onto most microscope stages. We leverage the computational fluidic dynamics techniques to calculate the forces based on the results obtained using our simple device. The EMT device is especially suitable to measure the traction forces at the dorsal side of a cell. As a proof of concept it was demonstrated that the EMT device could be applied to measure the dorsal traction forces exerted via the CD80-CTLA4 bond in metastatic cancer cells...
December 2018: BioTechniques
Filippo Piccinini, Ilaria De Santis, Alessandro Bevilacqua
No abstract text is available yet for this article.
December 2018: BioTechniques
Sean R Koebley, Jason Reed
No abstract text is available yet for this article.
November 2018: BioTechniques
Alistair R McTaggart, Tuan A Duong, Vang Quy Le, Louise S Shuey, Werner Smidt, Sanushka Naidoo, Michael J Wingfield, Brenda D Wingfield
It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq...
November 2018: BioTechniques
Bradford W Daigneault, Marcela Vilarino, Sandeep K Rajput, Tristan Frum, George W Smith, Pablo J Ross
CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection...
November 2018: BioTechniques
Scott G Canfield, Gyuhyung Jin, Sean P Palecek, Tori Sampsell
Cell culture is a vital component of laboratories throughout the scientific community, yet the absence of standardized protocols and documentation practice challenges laboratory efficiency and scientific reproducibility. We examined the effectiveness of a cloud-based software application, CultureTrax® as a tool for standardizing and transferring a complex cell culture protocol. The software workflow and template were used to electronically format a cardiomyocyte differentiation protocol and share a digitally executable copy with a different lab user...
November 2018: BioTechniques
Abigail Sawyer, Joseph Martin
Abigail Sawyer and Joseph Martin investigate how microscopy is evolving in order for cells and processes to be better visualized in their native environments.
November 2018: BioTechniques
Megan M Thompson, Estelle M Hrabak
Whatman FTA® Cards are a fast and efficient method for capturing and storing nucleic acids but can be cost-prohibitive for large numbers of samples. We developed a method that substitutes a readily-available cellulose matrix and homemade washing buffer for commercial FTA® Cards and FTA® Purification Reagent. This method is suitable for long-term storage of DNA from many plant species prior to PCR.
November 2018: BioTechniques
Mark F Kavlick
Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested. It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. The IPC was used in a hydrolysis probe qPCR assay; however, it may be adaptable for other applications such as intercalating dye qPCR, PCR, isothermal amplification and reverse transcription-PCR...
November 2018: BioTechniques
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