Most read Protocols
Protein Import Assay into Mitochondria Isolated from Human Cells
Authors:  Lena M. Murschall, Esra Peker, Thomas MacVicar, Thomas Langer and Jan Riemer, date: 06/20/2021, view: 1168, Q&A: 0

Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed.

Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening
Authors:  Henrik Renner, Mandy Otto, Martha Grabos, Hans R. Schöler and Jan M. Bruder, date: 06/20/2021, view: 1108, Q&A: 1

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues (“organoids”), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells

Tracking the Subcellular Localization of Surface Proteins in Staphylococcus aureus by Immunofluorescence Microscopy
Authors:  Salvatore J. Scaffidi, Mac A. Shebes and Wenqi Yu, date: 05/20/2021, view: 2976, Q&A: 0

Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment,

In vitro and In vivo CD8+ T Cell Suppression Assays
Authors:  Lu Xie, Guihuan Liu, Yanjun Liu and Yuming Yu, date: 05/20/2021, view: 2138, Q&A: 0

CD8+CD28− T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells. 3H-thymidine is a classical immunological

Intracellular IRF5 Dimerization Assay
Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 1915, Q&A: 0

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

A New Method for Studying RNA-binding Proteins on Specific RNAs
Authors:  Weiping Sun, Ziheng Zhang, Ji-Long Liu and Min Zhuang, date: 05/20/2021, view: 4237, Q&A: 0

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT.

Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins
Authors:  Rezwan Siddiquee and Ann H Kwan, date: 05/20/2021, view: 2100, Q&A: 0

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins)

FACS Enrichment of Total Interstitial Cells and Fibroblasts from Adult Mouse Ventricles
Authors:  Nona Farbehi, Vaibhao Janbandhu, Robert E. Nordon and Richard P. Harvey, date: 05/20/2021, view: 1203, Q&A: 0

Besides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac

Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase
Authors:  Abhishek Chhetri, Anna Loksztejn and Kenichi Yokoyama, date: 04/20/2021, view: 1850, Q&A: 0

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely

AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells
Authors:  Nathalie Bouquier, Enora Moutin, Julie Perroy and Vincent Ollendorff, date: 04/20/2021, view: 1343, Q&A: 0

Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases.

Computational Analysis and Phylogenetic Clustering of SARS-CoV-2 Genomes
Authors:  Bani Jolly and Vinod Scaria, date: 04/20/2021, view: 2336, Q&A: 0

COVID-19, the disease caused by the novel SARS-CoV-2 coronavirus, originated as an isolated outbreak in the Hubei province of China but soon created a global pandemic and is now a major threat to healthcare systems worldwide. Following the rapid human-to-human transmission of the infection, institutes around the world have made efforts to generate

Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
Authors:  Fernando L. Gordillo Altamirano and Jeremy J. Barr, date: 04/20/2021, view: 2643, Q&A: 1

Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal

A Robust Mammary Organoid System to Model Lactation and Involution-like Processes
Authors:  Elsa Charifou, Jakub Sumbal, Zuzana Koledova, Han Li and Aurélie Chiche, date: 04/20/2021, view: 2381, Q&A: 0

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding

Production of Phenotypically Uniform Human Cerebral Organoids from Pluripotent Stem Cells
Authors:  Adam Sivitilli, Parisa Ghiasi and Liliana Attisano, date: 04/20/2021, view: 2005, Q&A: 0

Recent advances in stem cell technology have allowed researchers to generate 3D cerebral organoids (COs) from human pluripotent stem cells (hPSCs). Indeed, COs have provided an unprecedented opportunity to model the developing human brain in a 3D context, and in turn, are suitable for addressing complex neurological questions by leveraging

A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 2607, Q&A: 0

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
Authors:  Gareth Howell and Robert-Jan Bleichrodt, date: 04/20/2021, view: 1655, Q&A: 0

Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here

Development of a Chemical Reproductive Aging Model in Female Rats

Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and

Single-unit Recording in Awake Behaving Non-human Primates
Authors:  Mengxi Yun, Masafumi Nejime and Masayuki Matsumoto, date: 04/20/2021, view: 809, Q&A: 0

Non-human primates (NHPs) have been widely used as a species model in studies to understand higher brain functions in health and disease. These studies employ specifically designed behavioral tasks in which animal behavior is well-controlled, and record neuronal activity at high spatial and temporal resolutions while animals are performing the

Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay
Authors:  Javier Aguilera and Jianjun Sun, date: 04/20/2021, view: 1319, Q&A: 0
The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across ...
A Method to Quantify Drosophila Behavioral Activities Induced by GABAA Agonist Muscimol
Author:  M. Rezaul Karim, date: 04/20/2021, view: 708, Q&A: 0

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria. As a potent GABAA receptor agonist, muscimol suppresses the activity of the central nervous system, reduces anxiety and induces sleep. We investigated the effects of muscimol on Drosophila behavior. Drosophila behavioral assays are powerful tools that are used to

Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
Authors:  Wyatt P. Bunner, Rachel Dodson, Robert M. Hughes and Erzsebet M. Szatmari, date: 04/20/2021, view: 876, Q&A: 0

Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the

Single or Repeated Ablation of Mouse Olfactory Epithelium by Methimazole
Authors:  Sofia Håglin, Staffan Bohm and Anna Berghard, date: 04/20/2021, view: 1886, Q&A: 0

Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature

Murine Monocyte and Macrophage Culture
Authors:  Simone M. Haag and Aditya Murthy, date: 03/20/2021, view: 3383, Q&A: 0

Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past

A Pipeline for Non-model Organisms for de novo Transcriptome Assembly, Annotation, and Gene Ontology Analysis Using Open Tools: Case Study with Scots Pine
Authors:  Gustavo T. Duarte, Polina Yu. Volkova and Stanislav A. Geras’kin, date: 02/05/2021, view: 2570, Q&A: 0

RNA sequencing (RNA-seq) has opened up the possibility of studying virtually any organism at the whole transcriptome level. Nevertheless, the absence of a sequenced and accurately annotated reference genome may be an obstacle for applying this technique to non-model organisms, especially for those with a complex genome. While de novo transcriptome

A Simple Microplate Assay for Reactive Oxygen Species Generation and Rapid Cellular Protein Normalization
Authors:  Neville S. Ng and Lezanne Ooi, date: 01/05/2021, view: 2305, Q&A: 0
2',7'-dichlorofluorescein (DCF) and derivatives are commonly used as fluorescent indicators of a broad spectrum of reactive oxygen species (ROS) generation in cell-based assays. However, there are numerous challenges inherent to the utilization of DCF probes for intracellular microscopic analysis, including bleaching and probe efflux. Plate ...
Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells
Authors:  Kun Ho Kim, Jiamin Qiu and Shihuan Kuang, date: 07/20/2020, view: 3397, Q&A: 0
The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and ...
Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
Authors:  Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 5253, Q&A: 0
Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...
Mouse Adipose Tissue Protein Extraction
Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 4535, 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 ...
Platelet Isolation and Activation Assays
Authors:  Laura C. Burzynski, Nicholas Pugh and Murray C.H. Clarke, date: 10/20/2019, view: 5529, Q&A: 0
Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, ...
Cylinder Test to Assess Sensory-motor Function in a Mouse Model of Parkinson’s Disease
Parkinson’s disease is a progressive neurodegenerative movement disorder that happens due to the loss of dopaminergic neurons in the substantia nigra. The deficiency of dopamine in the basal nuclei drives cardinal motor symptoms such as bradykinesia and hypokinesia. The current protocol describes the cylinder test, which is a relatively simple ...
Morphological Evaluation of Wound Healing Events in the Excisional Wound Healing Model in Rats
Skin wound healing is a complex process involving different events such as blood coagulation, inflammation, new blood vessels formation, and extracellular matrix deposition. These events can be observed by using histology techniques. However, the lack of the standardization of such parameters impacts on the reproducibility of results. Here, we ...
Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide
Authors:  Anthony Yammine, Jinlong Gao and Ann H. Kwan, date: 06/05/2019, view: 12781, Q&A: 0
Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. ...
Detection of Antigen-specific T cells in Spleens of Vaccinated Mice Applying 3[H]-Thymidine Incorporation Assay and Luminex Multiple Cytokine Analysis Technology
Authors:  Maria Agallou and Evdokia Karagouni, date: 06/05/2019, view: 7137, Q&A: 0
For many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. Here, we describe a protocol for the analysis of vaccine-induced antigen-specific immune responses. For this purpose, cells of whole spleens obtained from vaccinated ...
Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method
Authors:  Shraddha Gang, Sheetal Sharma, Meenu Saraf, Martin Buck and Jörg Schumacher, date: 05/05/2019, view: 15544, Q&A: 1
Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a ...
Assessing Spatial Working Memory Using the Spontaneous Alternation Y-maze Test in Aged Male Mice
Authors:  Emily A.K. Prieur and Nafisa M. Jadavji, date: 02/05/2019, view: 12685, Q&A: 0
The global population is aging and the prevalence of age-related diseases, such as Alzheimer's disease and vascular dementia is increasing. Understanding functional impairments and disease processes is of vital importance in order to develop effective therapeutics. Using the natural exploratory behavior of mice, the spontaneous alternation y-maze ...
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: 15307, 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: 23820, 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: 21300, 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 ...
Generation of Stable Expression Mammalian Cell Lines Using Lentivirus
Authors:  Neha Tandon, Kaushik N. Thakkar, Edward L LaGory, Yu Liu and Amato J Giaccia, date: 11/05/2018, view: 11946, Q&A: 1
Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 ...
Assessing Membrane Fluidity and Visualizing Fluid Membrane Domains in Bacteria Using Fluorescent Membrane Dyes
Authors:  Michaela Wenzel, Norbert O. E. Vischer, Henrik Strahl and Leendert W. Hamoen, date: 10/20/2018, view: 6970, Q&A: 0
Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, ...
Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
Author:  Sébastien Gillotin, date: 10/05/2018, view: 13519, Q&A: 5
Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes ...
Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen
Authors:  Fabian Tetzlaff and Andreas Fischer, date: 09/05/2018, view: 11962, 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: 57326, 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 ...
Microbial Mutagenicity Assay: Ames Test
Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 26433, 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: 17204, Q&A: 2
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 ...
Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining
Authors:  Janina R. Juengert, Stephanie Bresan and Dieter Jendrossek, date: 03/05/2018, view: 11661, Q&A: 0
Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence ...
MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
Authors:  Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 18211, Q&A: 0
Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...
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: 13221, 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 ...
Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)
Authors:  Rajneesh, Jainendra Pathak, Ananya Chatterjee, Shailendra P. Singh and Rajeshwar P. Sinha, date: 09/05/2017, view: 16865, Q&A: 0
Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ...
Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
Authors:  Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 30583, Q&A: 0
Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...
Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique
Membrane damage is a hallmark of both biotic and abiotic stress responses. The membrane determines the ability of a cell to sustain altered environmental conditions and hence can be used as a biomarker to assess stress-induced cell damage or death. We present an easy, quick, cost-effective, staining and spectrophotometric method to assess membrane ...
Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Author:  Oliver Wirths, date: 08/05/2017, view: 21732, 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: 26196, 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 Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea
Authors:  Giovanna Salbitani, Claudia Bottone and Simona Carfagna, date: 07/05/2017, view: 16484, Q&A: 3
Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved ...
Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
Authors:  Florian Hahn, Marion Eisenhut, Otho Mantegazza and Andreas P. M. Weber, date: 07/05/2017, view: 19022, Q&A: 1
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing ...
Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis
Authors:  Feng Du, Xiaodi Zhao and Daiming Fan, date: 06/20/2017, view: 23898, Q&A: 0
Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. After two weeks of incubation, the number of ...
Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana
Authors:  Takushi Hachiya and Yuki Okamoto, date: 05/20/2017, view: 15643, Q&A: 1
Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for ...
In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems
Authors:  Hong-Ru Chen and Trai-Ming Yeh, date: 05/05/2017, view: 20991, Q&A: 2
Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay ...
Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
Authors:  Valentina La Verde, Paola Dominici and Alessandra Astegno, date: 04/20/2017, view: 26659, 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 ( ...
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: 18412, 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 ...
Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
Authors:  Alaina Puleo, Chantia Carroll, Holden Maecker and Rohit Gupta, date: 01/20/2017, view: 30943, Q&A: 0
Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that ...
Dot Blot Analysis of N6-methyladenosine RNA Modification Levels
Authors:  Lisha Shen, Zhe Liang and Hao Yu, date: 01/05/2017, view: 21303, Q&A: 3
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 ...
Plant Tissue Trypan Blue Staining During Phytopathogen Infection
In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than ever. The protocol is applied specifically to detect cell death on Arabidopsis ...
Protocol for Molecular Dynamics Simulations of Proteins
Authors:  MNV Prasad Gajula, Anuj Kumar and Johny Ijaq, date: 12/05/2016, view: 20173, Q&A: 0
Molecular dynamics (MD) simulations have become one of the most important tools in understanding the behavior of bio-molecules on nanosecond to microsecond time scales. In this protocol, we provide a general approach and standard setup protocol for MD simulations by using the Gromacs MD suite.
Sucrose Preference Test to Measure Anhedonic Behaviour in Mice
Authors:  Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 26784, 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 ...
Light/Dark Transition Test to Assess Anxiety-like Behavior in Mice
Authors:  Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 17843, Q&A: 0
The light/dark transition test (LDT) is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in response to mild stressors, such as novel environment and light. This test is also sensitive to ...
Isolation of Intestinal Mesenchymal Cells from Adult Mice
Authors:  Vasiliki Koliaraki and George Kollias, date: 09/20/2016, view: 18319, Q&A: 0
During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue ...
Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test
Authors:  Jeremy Ferrier, Fabien Marchand and David Balayssac, date: 09/20/2016, view: 19668, Q&A: 0
Chronic pain is one of the most debilitating conditions, affecting one out of five people worldwide. Preclinical models in rodents represent a valuable tool for the study of pathophysiological mechanisms and the discovery of new analgesic drugs. However, pain evaluation in rodents is rather challenging. Altered response to mechanical or thermal ...
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: 46247, 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 ...
Rust Removal Experiments
Authors:  Mahmoud Kamal Ahmadi and Blaine A. Pfeifer, date: 04/05/2016, view: 19774, Q&A: 0
Iron oxidation, known as rust formation, causes enormous loss in term of property damages and associated economic risks. Depending on the degree of formation, rust consists of several layers of iron in different oxidation states. The brownish top layer is mostly iron (III) oxide-hydroxide [FeO(OH), Fe(OH)3] while the deepest black ...
In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells
Over the past decade, in vivo bioluminescent imaging has emerged as a non-invasive and sensitive tool for studying ongoing biological processes within living organisms (Contag et al., 1997; Contag et al., 1998). Based on the detection and quantitation of the photons produced by the oxidation of luciferin by luciferase ...
Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
Authors:  Kristofor K. Ellestad and Colin C. Anderson, date: 01/05/2016, view: 25776, Q&A: 0
Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately ...
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: 38426, Q&A: 2
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 ...
Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria
Authors:  Tomáš Zavřel, Maria A. Sinetova and Jan Červený, date: 05/05/2015, view: 28312, Q&A: 2
This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to ...
Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii
Authors:  Ming-Feng Lin, Yun-You Lin and Chung-Yu Lan, date: 12/05/2014, view: 43601, Q&A: 3
Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an ...
Analysis of Intestinal Permeability in Mice
Authors:  Jalaj Gupta and Angel R. Nebreda, date: 11/20/2014, view: 40533, Q&A: 7
The intestinal epithelial layer serves as a barrier against pathogens and ingested toxins, which are present in the lumen of the intestine. The importance of the intestinal epithelial barrier is emphasized by the alterations in paracellular permeability and tight junction functions observed in inflammatory bowel disease (IBD) and colon cancer.
Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses
Authors:  Anna Sloutskin and Ronald S. Goldstein, date: 11/20/2014, view: 38191, 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.
Extraction of Total Proteins from Rice Plant
Authors:  Da-Gin Lin and Chang-Sheng Wang, date: 11/05/2014, view: 37806, Q&A: 1
This protocol provides an efficient method for preparation of high-quality proteins from rice leaves and grains. The method involves phenol extraction to separate proteins from the non-protein components such as polysaccharides, lipids and phenolic compounds that are commonly enriched in plant tissues. Following isolation, proteins are ...
Dimethylmethylene Blue Assay (DMMB)
Authors:  Vivien Jane Coulson- Thomas and Tarsis Ferreira Gesteira, date: 09/20/2014, view: 50539, Q&A: 8
Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, ...
Elevated Plus Maze Test to Assess Anxiety-like Behavior in the Mouse
Authors:  Luciana M. Leo and Fabricio A. Pamplona, date: 08/20/2014, view: 25373, Q&A: 0
The elevated plus maze task is a simple method to assess anxiety-like behaviors in rodents. This version describes the procedure used in mice. However, the protocol may also be applied to rats, considering a proportionally larger apparatus (arms: 10 x 50 cm; height: 55 cm). Briefly, the test is performed on a plus-shaped apparatus with two open ...
Wnt Reporter Activity Assay
Author:  Chen Zhao, date: 07/20/2014, view: 30546, Q&A: 2
This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt ...
LT-HSC Methylcellulose Assay
Author:  Marc A. Kerenyi, date: 03/05/2014, view: 19978, Q&A: 0
Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter ...
ADCC Assay Protocol
Authors:  Vikram Srivastava, Zheng Yang, Ivan Fan Ngai Hung, Jianqing Xu, Bojian Zheng and Mei-Yun Zhang, date: 01/20/2014, view: 34780, Q&A: 0
Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections and cancers in vivo. Here we developed a flow cytometry based protocol for ADCC assay using human ...
In vitro Protein Ubiquitination Assays
Authors:  Qingzhen Zhao and Qi Xie, date: 10/05/2013, view: 40516, 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 ...
Blood AST, ALT and UREA/BUN Level Analysis
Authors:  Yuh-Pyng Sher and Mien-Chie Hung, date: 10/05/2013, view: 31996, Q&A: 0
AST (aspartate aminotransferase; GOT, glutamate oxalacetate transaminase) and ALT (alanine aminotransferase; GPT, glutamate pyruvate transaminase) are sensitive indicators to monitor the liver function under drugs treatment or with acute viral hepatitis. The elevated AST and ALT values in the blood sample indicate liver damage or injury. The ...
Neutral Comet Assay
Authors:  Elisa Boutet-Robinet, Didier Trouche and Yvan Canitrot, date: 09/20/2013, view: 31556, Q&A: 1
The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding ...
Seed Germination and Viability Test in Tetrazolium (TZ) Assay
Authors:  Pooja Verma and Manoj Majee, date: 09/05/2013, view: 55162, Q&A: 9
Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble formazan by hydrogen ...
Polysome Profiling Analysis
Authors:  Masahiro Morita, Tommy Alain, Ivan Topisirovic and Nahum Sonenberg, date: 07/20/2013, view: 42465, Q&A: 2
Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.
Flow Cytometric Detection of Reactive Oxygen Species
Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions ...
Isolation of Human PBMCs
Authors:  Santosh K Panda and Balachandran Ravindran, date: 02/05/2013, view: 84750, Q&A: 8
Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque.
In vitro Culture of Human PBMCs
Authors:  Santosh K Panda and Balachandran Ravindran, date: 02/05/2013, view: 100211, Q&A: 11
Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) ...
Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)
Authors:  Rabii Ameziane-El-Hassani and Corinne Dupuy, date: 01/05/2013, view: 78313, Q&A: 3
Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants systems. In several diseases (cancer, ...
Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves
Authors:  Arsalan Daudi and Jose A. O’Brien, date: 09/20/2012, view: 70457, Q&A: 22
In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al. ...
Senescence Associated β-galactosidase Staining
Authors:  Michael Eccles and Caiyun Grace Li, date: 08/20/2012, view: 61160, Q&A: 15
Detection of senescent cells using a cytochemical assay was first described in 1995 (Dimri et al., 1995). The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity (Kurz et al., 2000). Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized ...
Propidium Iodide Staining of Cells for FACS Analysis
Author:  Hui Zhu, date: 06/05/2012, view: 51582, Q&A: 2
Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA ...
Clonogenic Assay
Author:  Xiaodong Yang, date: 05/20/2012, view: 89285, Q&A: 7
Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing ...
Scratch Wound Healing Assay
Author:  Yanling Chen, date: 03/05/2012, view: 114791, 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” ...
Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)
Author:  Ran Chen, date: 02/05/2012, view: 62500, Q&A: 15
Bone marrow derived macrophages are a type of white blood cell that can be isolated from mammalian bone marrow. In this protocol, a method is described in which bone marrow cells are isolated from mouse leg bones (femur and tibia), and then differentiated to bone marrow-derived macrophages in approximately 10-day culture using L929-conditioned ...

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.