Most read Protocols
Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish
Authors:  Massimo Ganassi, Peter S. Zammit and Simon M. Hughes, date: 09/05/2021, view: 2337, Q&A: 0

Skeletal muscles generate force throughout life and require maintenance and repair to ensure efficiency. The population of resident muscle stem cells (MuSCs), termed satellite cells, dwells beneath the basal lamina of adult myofibres and contributes to both muscle growth and regeneration. Upon exposure to activating signals, MuSCs proliferate to

Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy
Authors:  Francesco S. Ruggeri, Alyssa M. Miller, Michele Vendruscolo and Tuomas P. J. Knowles, date: 08/20/2021, view: 5126, Q&A: 0

The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into

Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
Authors:  Patrick J. Shilling and Daniel O. Daley, date: 08/20/2021, view: 2407, Q&A: 0

pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used

CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes
Authors:  Masahito Yamagata and Joshua R. Sanes, date: 08/05/2021, view: 2217, Q&A: 0

The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes

GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging)

Isolation of Microglia and Analysis of Protein Expression by Flow Cytometry: Avoiding the Pitfall of Microglia Background Autofluorescence
Authors:  Jeremy C. Burns, Richard M. Ransohoff and Michaël Mingueneau, date: 07/20/2021, view: 2228, Q&A: 0

Microglia are a unique type of tissue-resident innate immune cell found within the brain, spinal cord, and retina. In the healthy nervous system, their main functions are to defend the tissue against infectious microbes, support neuronal networks through synapse remodeling, and clear extracellular debris and dying cells through phagocytosis. Many

Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures
Authors:  Gunhyoung Lim and Sungchul Hohng, date: 07/05/2021, view: 2044, Q&A: 0

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop

Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM)
Authors:  Simao Coelho, Jongho Baek, J. Justin Gooding and Katharina Gaus, date: 07/05/2021, view: 4949, Q&A: 0

The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision.

Micrografting in Arabidopsis Using a Silicone Chip
Authors:  Hiroki Tsutsui, Yaichi Kawakatsu and Michitaka Notaguchi, date: 06/20/2021, view: 2398, Q&A: 1

The micrografting technique in the model plant Arabidopsis has been widely used in the field of plant science. Grafting experiments have demonstrated that signal transductions are systematically regulated in many plant characteristics, including defense mechanisms and responses to surrounding environments such as soil and light conditions.

Mouse Adipose Tissue Protein Extraction
Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 6371, Q&A: 0
As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...
Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe
Authors:  Farzane Sivandzade, Aditya Bhalerao and Luca Cucullo, date: 01/05/2019, view: 18308, Q&A: 0
In recent years, fluorescent dyes have been frequently used for monitoring mitochondrial membrane potential to evaluate mitochondrial viability and function. However, the reproducibility of the results across laboratories strongly depends upon following well validated and reliable protocols along with the appropriate controls. Herein, we provide a ...
Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells
Authors:  Gal Haimovich and Jeffrey E. Gerst, date: 11/05/2018, view: 25882, Q&A: 1
Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects ...
mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells
Authors:  Madara Ratnadiwakara and Minna-Liisa Änkö, date: 11/05/2018, view: 24069, Q&A: 2
Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs ...
Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen
Authors:  Fabian Tetzlaff and Andreas Fischer, date: 09/05/2018, view: 13490, Q&A: 0
Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of ...
Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
Authors:  Birte Plitzko and Sandra Loesgen, date: 05/20/2018, view: 60545, Q&A: 1
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual ...
Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
Authors:  Stefanie N. Bernas, Odette Leiter, Tara L. Walker and Gerd Kempermann, date: 11/05/2017, view: 14743, Q&A: 0
There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation ...
Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Author:  Oliver Wirths, date: 08/05/2017, view: 23379, Q&A: 1
The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western ...
Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader
Authors:  Magdiel Martínez, Namyr A. Martínez and Walter I. Silva, date: 07/20/2017, view: 27891, Q&A: 1
Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured ...
Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
Authors:  Valentina La Verde, Paola Dominici and Alessandra Astegno, date: 04/20/2017, view: 28262, Q&A: 0
Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties ( ...
Sucrose Preference Test to Measure Anhedonic Behaviour in Mice
Authors:  Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 28784, Q&A: 0
The sucrose preference test (SPT) is a reward-based test, used as in indicator of anhedonia. Anhedonia, or the decreased ability to experience pleasure, represents one of the core symptoms of depression. Rodents are born with an interest in sweet foods or solutions. Reduced preference for sweet solution in SPT represents anhedonia, while this ...

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