Most Read —BIO-PROTOCOL

Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA

Authors: Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 514, Q&A: 0

Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...

Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow

Authors: Alexander Dupuy, Lining Arnold Ju and Freda H Passam, date: 03/20/2019, view: 3750, Q&A: 0

Microfluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation. Furthermore, microfluidic chips offer the benefits of ex vivo testing of platelet adhesion using small amounts of blood or purified platelets. ...

Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos

Authors: Rachna Narayanan and Andrew C. Oates, date: 03/20/2019, view: 3321, Q&A: 0

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to ...

Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) into an Effective Model of Forebrain Neural Progenitor Cells and Mature Neurons

Authors: Scott Bell, Nuwan C. Hettige, Heika Silveira, Huashan Peng, Hanrong Wu, Malvin Jefri, Lilit Antonyan, Ying Zhang, Xin Zhang and Carl Ernst, date: 03/05/2019, view: 3485, Q&A: 0

Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells that can be generated from somatic cells, and provide a way to model the development of neural tissues in vitro. One particularly interesting application of iPSCs is the development of neurons analogous to those found in the human forebrain. Forebrain neurons play a central ...

Induced Germinal Center B Cell Culture System

Authors: Kei Haniuda and Daisuke Kitamura, date: 02/20/2019, view: 3454, Q&A: 0

The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo ...

In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells

Authors: Nataša Savić, Femke CAS Ringnalda, Christian Berk, Katja Bargsten, Jonathan Hall, Martin Jinek and Gerald Schwank, date: 01/05/2019, view: 3314, Q&A: 0

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating ...

Evaluation of Anticancer activity of Silver Nanoparticles on the A549 Human Lung Carcinoma Cell Lines through Alamar Blue Assay

Authors: Nikita Sharma, Geeta Arya, R. Mankamna Kumari, Nidhi Gupta and Surendra Nimesh, date: 01/05/2019, view: 4220, Q&A: 0

Silver nanoparticles have been widely studied to possess antimicrobial as well as anticancer activity, and have found its applications in various fields including pharmaceutical industry, diagnostics, drug delivery, food industry, and others. For this purpose, several cell proliferation assays are widely used for the evaluation of anticancer ...

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: 5707, 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: 10128, 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 ...

Platelet Migration and Bacterial Trapping Assay under Flow

Authors: Shuxia Fan, Michael Lorenz, Steffen Massberg and Florian Gaertner, date: 09/20/2018, view: 2436, Q&A: 0

Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing ...

Cell Synchronization by Double Thymidine Block

Authors: Guo Chen and Xingming Deng, date: 09/05/2018, view: 16076, Q&A: 1

Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at ...

Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution

Author: Andrea Saponaro, date: 08/05/2018, view: 10896, Q&A: 0

This protocol can be applied to analyze the direct interaction between a soluble protein and a target ligand molecule using Isothermal Titration Calorimetry (ITC, Malvern). ITC allows the biophysical characterization of binding between label-free, non-immobilized and in-solution biomolecules by providing the stoichiometry of the interaction, the ...

In vitro Osteoclastogenesis Assays Using Primary Mouse Bone Marrow Cells

Authors: Xueqian Zhuang and Guohong Hu, date: 06/05/2018, view: 4247, Q&A: 1

Osteoclasts are a group of bone-absorbing cells to degenerate bone matrix and play pivotal roles in bone growth and homeostasis. The unbalanced induction of osteoclast differentiation (osteoclastogenesis) in pathological conditions, such as osteoporosis, arthritis and skeleton metastasis of cancer, causes great pain, bone fracture, hypercalcemia ...

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: 30568, Q&A: 0

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 ...

Microbial Mutagenicity Assay: Ames Test

Authors: Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 16846, Q&A: 2

The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...

Quantification of Plant Cell Death by Electrolyte Leakage Assay

Authors: Noriyuki Hatsugai and Fumiaki Katagiri, date: 03/05/2018, view: 9394, Q&A: 1

We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored ...

Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq

Authors: Lorrayne Serra, Dennis Z. Chang, Marissa Macchietto, Katherine Williams, Rabi Murad, Dihong Lu, Adler R. Dillman and Ali Mortazavi, date: 02/20/2018, view: 9153, Q&A: 3

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as ...

Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)

Authors: Dania M. Malik, Seth Rhoades and Aalim Weljie, date: 02/05/2018, view: 3779, Q&A: 0

Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a ...

Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes

Authors: Takeshi Katsuda, Kazunori Hosaka and Takahiro Ochiya, date: 01/20/2018, view: 4743, Q&A: 0

Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture ...

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

Authors: Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 14223, Q&A: 1

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the ...

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID

Authors: Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 6985, 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 ...

Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs

Authors: Vincent Loubiere, Anna Delest, Bernd Schuettengruber, Anne-Marie Martinez and Giacomo Cavalli, date: 06/05/2017, view: 6811, Q&A: 1

Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological ...

Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining

Authors: Chao Wang, Feng Yue and Shihuan Kuang, date: 05/20/2017, view: 12957, Q&A: 1

Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber ...

Murine Bronchoalveolar Lavage

Authors: Fan Sun, Gutian Xiao and Zhaoxia Qu, date: 05/20/2017, view: 14240, Q&A: 1

A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, ...

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery

Authors: Giel Vandemoortele, Delphine De Sutter and Sven Eyckerman, date: 04/05/2017, view: 13043, Q&A: 0

The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair ...

Polysome Fractionation to Analyze mRNA Distribution Profiles

Authors: Amaresh C. Panda, Jennifer L. Martindale and Myriam Gorospe, date: 02/05/2017, view: 15212, Q&A: 4

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that ...

Dot Blot Analysis of N⁶-methyladenosine RNA Modification Levels

Authors: Lisha Shen, Zhe Liang and Hao Yu, date: 01/05/2017, view: 13888, Q&A: 2

N⁶-methyladenosine (m⁶A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m⁶A can be detected by several methods, such as dot blot analysis using specific m⁶A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al ...

Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation

Authors: Esteban A. Orellana and Andrea L. Kasinski, date: 11/05/2016, view: 33534, Q&A: 0

The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be ...

BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry

Authors: Bo Qiu and M. Celeste Simon, date: 09/05/2016, view: 34324, Q&A: 0

Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis ...

Hemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies

Authors: Ying Wu, MyungSam Cho, David Shore, Manki Song, JungAh Choi, Tao Jiang, Yong-Qiang Deng, Melissa Bourgeois, Lynn Almli, Hua Yang, Li-Mei Chen, Yi Shi, Jianxu Qi, An Li, Kye Sook Yi, MinSeok Chang, Jin Soo Bae, HyunJoo Lee, JiYoung Shin, James Stevens, SeoungSuh Hong, Cheng-Feng Qin, George F. Gao, Shin Jae Chang and Ruben O. Donis, date: 06/05/2016, view: 22025, Q&A: 0

Heamagglutination is inhibited when antibodies are present because antibodies to the influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. Human monoclonal antibodies generated from single human B cells were tested to characterize their ...

Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells

Authors: Michael Peter Smith, Helen Young, Adam Hurlstone and Claudia Wellbrock, date: 11/05/2015, view: 26680, Q&A: 1

Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combing both targeted- and immuno-therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying ...

Analysis of Protein Stability by the Cycloheximide Chase Assay

Authors: Shih-Han Kao, Wen-Lung Wang, Chi-Yuan Chen, Yih-Leong Chang, Yi-Ying Wu, Yi-Ting Wang, Shu-Ping Wang, Alexey I Nesvizhskii, Yu-Ju Chen, Tse-Ming Hong and Pan-Chyr Yang, date: 01/05/2015, view: 48645, Q&A: 1

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a ...

Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses

Authors: Anna Sloutskin and Ronald S. Goldstein, date: 11/20/2014, view: 30135, Q&A: 0

Titration of viral stocks is a critical process before any experimental use of the virus. Here we describe an infectious focus assay for several alphaherpesviruses, a titration method for fluorescently labeled viruses, based on the original plaque assay. In addition, the calculation of multiplicity of infection (MOI) is presented.

Novel Object Recognition for Studying Memory in Mice

Authors: Tzyy-Nan Huang and Yi-Ping Hsueh, date: 10/05/2014, view: 24621, Q&A: 0

Memory tests are important indexes of the brain functions for rodents behavior assay. Many memory tasks require external forces (e.g. electric shocks) or intrinsic forces (e.g. hunger and thirsty) to trigger the responses. Under those conditions, rodents are under stresses, such as pain, tired, malnutrition or dehydration, which ...

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System

Authors: Kabin Xie, Bastian Minkenberg and Yinong Yang, date: 09/05/2014, view: 23683, Q&A: 0

RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 ...

In vitro Protein Ubiquitination Assays

Authors: Qingzhen Zhao and Qi Xie, date: 10/05/2013, view: 32382, Q&A: 0

Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or ...

Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation

Authors: Ying Liu, Keqiang Chen, Chunyan Wang, Wanghua Gong, Teizo Yoshimura, Ji Ming Wang and Mingyong Liu, date: 09/05/2013, view: 31156, Q&A: 0

A hallmark of cancer-associated inflammation is the infiltration of leukocytes into tumors, which is believed to be recruited by chemokines. Some infiltrating leukocytes such as macrophages often promote tumor growth by producing growth-inducing and angiogenic factors. Here, we described a method of isolating tumor-infiltrating leukocytes with ...

ImmunoPrecipitation of Nuclear Protein with Antibody Affinity Columns

Authors: Saïda Dadi, Dominique Payet-Bornet and Pierre Ferrier, date: 02/05/2013, view: 26917, Q&A: 1

Co-Immunoprecipitation (Co-IP) is the method used to pull down protein partners of a protein of interest using an antibody that specifically binds to this specific protein in order to test protein-protein interaction. “Pulled down” proteins can be analyzed by western blot for suspected protein partner, or by Mass spectrometry for high throughput ...

Scratch Wound Healing Assay

Author: Yanling Chen, date: 03/05/2012, view: 101243, Q&A: 6

The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer is created by scratching, and the “healing” ...

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen, date: 02/20/2012, view: 60464, Q&A: 6

Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous ...

Protocol for Measuring Free (Low-stress) Exploration in Rats