Most read Protocols
Whole-genome Methylation Analysis of APOBEC Enzyme-converted DNA (~5 kb) by Nanopore Sequencing

In recent years, DNA methylation research has been accelerated by the advent of nanopore sequencers. However, read length has been limited by the constraints of base conversion using the bisulfite method, making analysis of chromatin content difficult. The read length of the previous method combining bisulfite conversion and long-read sequencing

Labeling Endogenous Proteins Using CRISPR-mediated Insertion of Exon (CRISPIE)
Authors:  Evan A. Wilson, Tianyi Mao and Haining Zhong, date: 03/05/2022, view: 5355, Q&A: 0

The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the

Labeling and Tracking Mitochondria with Photoactivation in Drosophila Embryos
Authors:  Sayali Chowdhary and Richa Rikhy, date: 03/05/2022, view: 2061, Q&A: 0

Mitochondria are relatively small, fragmented, and abundant in the large embryos of Drosophila, Xenopus and zebrafish. It is essential to study their distribution and dynamics in these embryos to understand the mechanistic role of mitochondrial function in early morphogenesis events. Photoactivation of mitochondrially tagged GFP (mito-PA-GFP) is

An in vitro Blood-brain Barrier Model to Study the Penetration of Nanoparticles
Authors:  Sezin Aday, Wen Li, Jeffrey M. Karp and Nitin Joshi, date: 02/20/2022, view: 6349, Q&A: 0

The blood-brain barrier (BBB), a crucial protection mechanism in the central nervous system (CNS), is a selective barrier comprised of endothelial cells. It hampers the development of therapeutic and diagnostic tools for neurological diseases due to the poor penetration of most of these agents. Rationally engineered nanoparticles (NP) can

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids
Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are ...
NBD-lipid Uptake Assay for Mammalian Cell Lines
Authors:  Sara Abad Herrera, Marta Grifell-Junyent and Thomas Günther Pomorski, date: 02/20/2022, view: 2277, Q&A: 0

All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their

Developing Clinically Relevant Acquired Chemoresistance Models in Epithelial Ovarian Cancer Cell Lines
Authors:  Priti S. Shenoy, Sourav Chakraborty, Snehal M. Gaikwad, Asmita Sakpal and Pritha Ray, date: 02/05/2022, view: 2079, Q&A: 0

Chemoresistance, the ability of cancer cells to overcome therapeutic interventions, is an area of active research. Studies on intrinsic and acquired chemoresistance have partly succeeded in elucidating some of the molecular mechanisms in this elusive phenomenon. Hence, drug-resistant cellular models are routinely developed and used to mimic the

Large-scale Analysis of Sleep in Zebrafish
Authors:  Daniel A. Lee, Grigorios Oikonomou and David A. Prober, date: 02/05/2022, view: 2410, Q&A: 0

Over the past decade, zebrafish have emerged as a powerful model for the study of vertebrate sleep and wake behaviors. Experimental evidence has demonstrated behavioral, anatomical, genetic, and pharmacological conservation of sleep between zebrafish and mammals, suggesting that discoveries in zebrafish can inform our understanding of mammalian

ATAC Sequencing Protocol For Cryopreserved Mammalian Cells
Authors:  Juan Manuel Caravaca, Monika Mehta, Sujatha Gowda and Bao Tran, date: 01/20/2022, view: 2341, Q&A: 0

ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful method to evaluate chromatin accessibility and nucleosome positioning at a genome-wide scale. This assay uses a hyperactive Tn5 transposase, to simultaneously cut open chromatin and insert adapter sequences. After sequencing, the reads generated

Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI
Authors:  Vinayak Sadasivam Tumuluri and Kayarat Saikrishnan, date: 01/05/2022, view: 2233, Q&A: 1

Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used

Total Nucleic Acid Extraction from Single Zebrafish Embryos for Genotyping and RNA-seq
Authors:  Neha Wali, Munise Merteroglu, Richard J. White and Elisabeth M. Busch-Nentwich, date: 01/05/2022, view: 2383, Q&A: 0

RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a

A Simple Technique for Direct Immobilization of Target Enzymes from Cell Lysates Based on the SpyTag/SpyCatcher Spontaneous Reaction
Authors:  Li-xi Cai, Yuan-qing Lin, Yun-meng Chu, Xiao-pin Chen, Li-xing Liu, Ming Zhang and Guang-ya Zhang, date: 01/05/2022, view: 2090, Q&A: 0

Many of the current methods for enzyme purification and immobilization suffer from several drawbacks, such as requiring tedious multistep procedures or long preparation, and being environmentally unfriendly, due to the chemicals and conditions involved. Thus, a simple technique for direct purification and immobilization of target enzymes from cell

Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
Authors:  Patrick J. Shilling and Daniel O. Daley, date: 08/20/2021, view: 5810, 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: 5587, 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)

Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation
Authors:  Jinyun Wu, Yuqi Cai, Xiaoyang Wu, Yue Ying, Yilin Tai and Miao He, date: 03/05/2021, view: 8321, Q&A: 0
Transcardiac perfusion with saline followed by 4% paraformaldehyde (PFA) is widely used to clear blood and preserve brain for immunostaining or in situ hybridization. PFA breaks into formaldehyde in solution, which cross-link protein and DNA molecules to preserve tissue and cell structure. Here we provide a step by step guide for ...
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: 65794, 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: 17480, 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 ...
Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
Authors:  Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 17466, Q&A: 0
Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...
Sucrose Preference Test to Measure Anhedonic Behaviour in Mice
Authors:  Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 32591, 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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