Nature Protocols- Issue - nature.com science feeds

Using the rat forced swim test to assess antidepressant-like activity in rodents

David A Slattery — 2012-05-03

Using the rat forced swim test to assess antidepressant-like activity in rodents

Nature Protocols 7, 1009 (2012). doi:10.1038/nprot.2012.044

Authors: David A Slattery & John F Cryan

The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test

Real-time detection of acetylcholine release from the human endocrine pancreas

Rayner Rodriguez-Diaz — 2012-05-03

Real-time detection of acetylcholine release from the human endocrine pancreas

Nature Protocols 7, 1015 (2012). doi:10.1038/nprot.2012.040

Authors: Rayner Rodriguez-Diaz, Robin Dando, Y Anthony Huang, Per-Olof Berggren, Stephen D Roper & Alejandro Caicedo

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In

Genome-wide copy number analysis of single cells

Timour Baslan — 2012-05-03

Genome-wide copy number analysis of single cells

Nature Protocols 7, 1024 (2012). doi:10.1038/nprot.2012.039

Authors: Timour Baslan, Jude Kendall, Linda Rodgers, Hilary Cox, Mike Riggs, Asya Stepansky, Jennifer Troge, Kandasamy Ravi, Diane Esposito, B Lakshmi, Michael Wigler, Nicholas Navin & James Hicks

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

Erdinc Sezgin — 2012-05-03

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

Nature Protocols 7, 1042 (2012). doi:10.1038/nprot.2012.059

Authors: Erdinc Sezgin, Hermann-Josef Kaiser, Tobias Baumgart, Petra Schwille, Kai Simons & Ilya Levental

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

David Rabuka — 2012-05-10

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

Nature Protocols 7, 1052 (2012). doi:10.1038/nprot.2012.045

Authors: David Rabuka, Jason S Rush, Gregory W deHart, Peng Wu & Carolyn R Bertozzi

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can

Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells

Jin Zhang — 2012-05-10

Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells

Nature Protocols 7, 1068 (2012). doi:10.1038/nprot.2012.048

Authors: Jin Zhang, Esther Nuebel, Dona R R Wisidagama, Kiyoko Setoguchi, Jason S Hong, Christine M Van Horn, Sarah S Imam, Laurent Vergnes, Cindy S Malone, Carla M Koehler & Michael A Teitell

Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required

Visualization of gene expression in whole mouse retina by in situ hybridization

Michael B Powner — 2012-05-10

Visualization of gene expression in whole mouse retina by in situ hybridization

Nature Protocols 7, 1086 (2012). doi:10.1038/nprot.2012.050

Authors: Michael B Powner, Kristis Vevis, Jenny A G McKenzie, Pranita Gandhi, Shalini Jadeja & Marcus Fruttiger

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is

Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization

Harita Rao — 2012-05-10

Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization

Nature Protocols 7, 1097 (2012). doi:10.1038/nprot.2012.046

Authors: Harita Rao, Arun A Tanpure, Anupam A Sawant & Seergazhi G Srivatsan

This protocol describes the detailed experimental procedure for the synthesis of an azide-modified uridine triphosphate analog and its effective incorporation into an oligoribonucleotide by in vitro transcription reactions. Furthermore, procedures for labeling azide-modified oligoribonucleotides post-transcriptionally with biophysical probes by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and

Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy

Shuang Wu — 2012-05-17

Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy

Nature Protocols 7, 1113 (2012). doi:10.1038/nprot.2012.056

Authors: Shuang Wu, Tobias I Baskin & Kimberly L Gallagher

Despite improvements in live imaging, fixation followed by embedding and sectioning for light or electron microscopy remains an indispensible approach in biology. During processing, small or delicate samples can be lost, damaged or poorly oriented. Here we present a protocol for overcoming these issues when,

Mussel micronucleus cytome assay

Claudia Bolognesi — 2012-05-17

Mussel micronucleus cytome assay

Nature Protocols 7, 1125 (2012). doi:10.1038/nprot.2012.043

Authors: Claudia Bolognesi & Michael Fenech

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection

Rafael Fridman — 2012-05-17

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection

Nature Protocols 7, 1138 (2012). doi:10.1038/nprot.2012.053

Authors: Rafael Fridman, Gabriel Benton, Irina Aranoutova, Hynda K Kleinman & R Daniel Bonfil

This protocol requires 2–4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells

Quan-Xiang Wei — 2012-05-17

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells

Nature Protocols 7, 1145 (2012). doi:10.1038/nprot.2012.042

Authors: Quan-Xiang Wei, Franciscus van der Hoeven, Monica Hollstein & Adam F Odell

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform'