1Department of Horticulture and Landscape Architecture, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, 4College of Environmental & Resource Science, Zhejiang University, 5Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, 6Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences
Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.
Published September 2, 2014. Keywords: Plant Biology, Aequorin, Case12, abiotic stress, heavy metal stress, copper ion, calcium imaging, Arabidopsis
1Department of Biological Sciences, Carnegie Mellon University
Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.
Published September 1, 2014. Keywords: Developmental Biology, Embryology, Patiria miniata, sea star, echinoderm, development, gene regulatory networks, microinjection, gene expression perturbation, antisense oligonucleotide, reporter expression
1Department of Biology, American University, 2Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH
The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.
Published September 1, 2014. Keywords: Cancer Biology, Angiogenesis, tube formation, fibroblast, endothelial cell, matrix, 3B-11, basement membrane extract, tubulogenesis
1Energy, Mining and Environment, National Research Council Canada
Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.
Published August 29, 2014. Keywords: Molecular Biology, Metagenomics, Bacteria, 16S ribosomal RNA gene, Amplicon sequencing, Next-generation sequencing, benchtop sequencers
1Department of Molecular Genetics, Weizmann Institute of Science
Isolated pancreatic acini retain their in vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.
Published August 28, 2014. Keywords: Cellular Biology, Cell biology, Exocrine secretion, Pancreatic acinar cells, Primary culture, Exocytosis, Actin, Lifeact
1LENS - European Laboratory for Non-linear Spectroscopy, University of Florence, 2Chemistry Research Laboratory, University of Oxford, 3Department of Biology, University of Florence, 4Department of Physics and Astronomy, University of Florence, 5National Institute of Optics-National Research Council, Italy, 6International Center of Computational Neurophotonics
Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.
Published August 27, 2014. Keywords: Bioengineering, Single molecule biophysics, Optical tweezers, fluorescence microscopy, DNA binding proteins, lactose repressor, microfluidics
1New Jersey Neuroscience Institute, JFK Medical Center, 2Department of Biomedical Engineering, Rutgers University, 3Department of Mechanical and Aerospace Engineering, Rutgers University
Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
Published August 27, 2014. Keywords: Bioengineering, bioengineering (general), Elastic, viscoelastic, bis-acrylamide, substrate, stiffness, dynamic, static, neuron, fibroblast, compliance, ECM, mechanobiology, tunable
1Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN)
This article describes microinjection and electroporation of mouse testis in vivo as a transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.
Published August 23, 2014. Keywords: Molecular Biology, electroporation, transfection, microinjection, testis, sperm, spermatogenesis, reproduction
1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University
Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations. However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.
Published August 23, 2014. Keywords: Molecular Biology, Electroporation, human embryonic stem cell, genome editing, reporter cell line, midbrain dopaminergic neurons
1Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, 2Center for Cardiovascular and Pulmonary Research, The Heart Center, The Research Institute at Nationwide Children's Hospital, 3Department of Pediatrics, The Ohio State University
The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.
Published August 21, 2014. Keywords: Cellular Biology, Heart valve, Valve Endothelial Cells (VEC), Fluorescence Activated Cell Sorting (FACS), Mouse, Embryo, Adult, GFP.