• Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Current Issue in 2019

Volume: 9, Issue: 18

Biochemistry

Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient Authors:  Julian M. Carosi, Kathryn J. Hattersly, Yi Cui, Zhe Yang, Rohan D. Teasdale and Timothy J. Sargeant, date: 09/20/2019, view: 99, Q&A: 0
The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or ...
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Cancer Biology

A Cell Culture Model that Mimics Physiological Tissue Oxygenation Using Oxygen-permeable Membranes Authors:  Chloe-Anne Martinez, Peter A. Cistulli and Kristina M. Cook, date: 09/20/2019, view: 78, Q&A: 0
Dissolved oxygen and its availability to cells in culture is an overlooked variable which can have significant consequences on experimental research outcomes, including reproducibility. Oxygen sensing pathways play key roles in cell growth and behavior and pericellular oxygen levels should be controlled when establishing in vitro models. ...
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SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks Authors:  Sunetra Roy and Katharina Schlacher, date: 09/20/2019, view: 51, Q&A: 0
The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. ...
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Cell Biology

Characterization of Biological Motion Using Motion Sensing Superpixels Authors:  Felix Y. Zhou, Carlos Ruiz-Puig, Richard P. Owen, Michael J. White, Jens Rittscher and Xin Lu, date: 09/20/2019, view: 40, Q&A: 0
Precise spatiotemporal regulation is the foundation for the healthy development and maintenance of living organisms. All cells must correctly execute their function in the right place at the right time. Cellular motion is thus an important dynamic readout of signaling in key disease-relevant molecular pathways. However despite the rapid ...
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Supported Cell Membrane Sheets to Monitor Protein Assembly Authors:  Simon Erlendsson, Thor Seneca Thorsen and Kenneth Lindegaard Madsen, date: 09/20/2019, view: 48, Q&A: 0
Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, ...
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Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal Mouse Macrophages by Flow Cytometry Authors:  Katharina Richard, Stefanie N. Vogel and Darren J. Perkins, date: 09/20/2019, view: 41, Q&A: 0
Internalization of cell surface Toll-like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.
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Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells Authors:  I-Ting Teng, Xiangning Bu and Inhee Chung, date: 09/20/2019, view: 50, Q&A: 0
Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have ...
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Immunology

Immunohistochemical Staining of CD8α in Diabetic Mouse Kidney Authors:  Ming-Zhi Zhang and Raymond C. Harris, date: 09/20/2019, view: 46, Q&A: 0
Immune cell infiltration, particularly cytotoxic CD8α lymphocyte infiltration, plays an important role in development of diabetic nephropathy. Although CD8α infiltration can be evaluated by its production of cytokines, its localization in the kidney is of particular importance. The current protocol describes CD8α immunostaining using a Vectastain ...
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Microbiology

Production of Quantum Dots-containing Influenza Virus Particles for Studying Viral Uncoating Processes Authors:  Chong Qin, Xiaowei Zhang and Zongqiang Cui, date: 09/20/2019, view: 36, Q&A: 0
The genome of influenza A virus (IAV) comprises eight pinlike genomic segments called vRNPs enclosed in viral capsid. During infection, uncoating is the key step for viral replication and represents an antiviral therapeutic target, but it is difficult to observe the transient and dynamic event in detail. Here, we report a protocol for production ...
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Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria Authors:  Lidia Delgado, Nicolás Baeza, Carla Pérez-Cruz, Carmen López-Iglesias and Elena Mercadé, date: 09/20/2019, view: 42, Q&A: 0
A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as ...
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Molecular Biology

A PhoA-STII Based Method for Efficient Extracellular Secretion and Purification of Fab from Escherichia coli Authors:  Ziyan Wang, Yang Gao, Manyu Luo, Cedric Cagliero, Hua Jiang, Yueqing Xie, Jianwei Zhu and Huili Lu, date: 09/20/2019, view: 36, Q&A: 0
In comparison with full-length IgGs, antigen binding fragments (Fabs) are smaller in size and do not require the complexed post-translational modification. Therefore, Fab can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, the disulfide-bonds containing exogenous protein, including ...
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In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression Authors:  Joanna Kud, Nejra Solo, Allan Caplan, Joseph C. Kuhl, Louise-Marie Dandurand and Fangming Xiao, date: 09/20/2019, view: 39, Q&A: 0
In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin ...
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Using indCAPS to Detect CRISPR/Cas9 Induced Mutations Authors:  Charles Hodgens, Zachary L. Nimchuk and Joseph J. Kieber, date: 09/20/2019, view: 38, Q&A: 0
Cleaved amplified polymorphic sequences (CAPS) assays are useful tools for detecting small mutations such as single nucleotide polymorphisms (SNPs) or insertion/deletions (indels) present in an amplified DNA fragment. A mutation that disrupts or creates a restriction site will prevent cleavage by a restriction enzyme, allowing discrimination of ...
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Neuroscience

Sciatic Nerve Cut and Repair Using Fibrin Glue in Adult Mice Authors:  Erica T. Akhter, Travis M. Rotterman, Arthur W. English and Francisco J. Alvarez, date: 09/20/2019, view: 47, Q&A: 0
Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to ...
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Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation Authors:  Mariko Kinoshita-Kawada, Hiroshi Hasegawa, Tsunaki Hongu, Shigeru Yanagi, Yasunori Kanaho, Ichiro Masai, Takayasu Mishima, Xiaoping Chen, Yoshio Tsuboi, Yi Rao, Junichi Yuasa-Kawada and Jane Y. Wu, date: 09/20/2019, view: 45, Q&A: 0
Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in ...
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Simple Protocol for Distinguishing Drug-induced Effects on Spatial Memory Acquisition, Consolidation and Retrieval in Mice Using the Morris Water Maze Authors:  Ariel K. Frame, Asad Lone, Richard A. Harris and Robert C. Cumming, date: 09/20/2019, view: 52, Q&A: 0
The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or duration of drug exposure when conducting the MWM assay; factors that can strongly influence the effect ...
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