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Cell Biology

Glioma Induction by Intracerebral Retrovirus Injection

Featured protocol,  Authors: Ravinder K Verma
Ravinder K Verma Affiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Bio-protocol author page: a4878
Fanghui Lu
Fanghui LuAffiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Bio-protocol author page: a4879
 and Qing Richard Lu
Qing Richard LuAffiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
For correspondence: richard.lu@cchmc.org
Bio-protocol author page: a4880
date: 7/20/2017, 22 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2404.

Brief version appeared in Cancer Cell, May 2016
Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide a better understanding in the mechanisms of gliomagenesis, but also facilitate the drug discovery for treating this deadly cancer. A retroviral vector system that expresses PDGFBB (Platelet-derived growth factor BB) and inactivates PTEN (Phosphatase and tensin homolog) and P53 tumor suppressors provides a rapid and efficient induction of glioma in mice with full penetrance. In this protocol, we describe a simple and practical method for inducing GBM formation by retrovirus injection in the murine brain. This system gives a spatial and temporal control over the induction of glioma and allows the assessment of therapeutic effects with a bioluminescent reporter.

Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs)

Featured protocol,  Authors: Wei Xu
Wei XuAffiliation 1: Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Affiliation 2: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
Bio-protocol author page: a4915
 and Chengbiao Wu
Chengbiao WuAffiliation: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
For correspondence: chw049@ucsd.edu
Bio-protocol author page: a4916
date: 7/20/2017, 16 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2413.

Brief version appeared in J Clin Invest, May 2016
The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) is believed to be involved in many disorders of the brain such as Alzheimer’s disease (AD), schizophrenia, sleep disorders and drug abuse (Mobley et al., 1986). Primary cultures of BFCNs will provide an important tool for studying the mechanism of these diseases. This protocol provides a detailed description of experimental procedures in establishing in vitro primary culture of rat embryonic BFCNs.

Non-radioactive LATS in vitro Kinase Assay

Featured protocol,  Authors: Audrey W. Hong
Audrey W. HongAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
Bio-protocol author page: a4827
 and Kun-Liang Guan
Kun-Liang GuanAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
For correspondence: kuguan@ucsd.edu
Bio-protocol author page: a868
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2391.

Brief version appeared in EMBO Rep, Jan 2017
This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Featured protocol,  Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

Brief version appeared in eLife, Jan 2017
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.

Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Featured protocol,  Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

Brief version appeared in Elife, Nov 2016
Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population. This protocol describes how to collect and germinate the spores of U. bromivora on plate in order to obtain strains derived from a single cell.

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Featured protocol,  Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

Brief version appeared in PLoS Pathog, Jun 2016
Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.

Plasmodium Sporozoite Motility on Flat Substrates

Featured protocol,  Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

Brief version appeared in PLoS Pathog, Jul 2016
Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster

Featured protocol,  Authors: Benjamin A. Eaton
Benjamin A. EatonAffiliation 1: Department of Cellular and Integrative Physiology, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Bio-protocol author page: a4872
 and Rebekah E. Mahoney
Rebekah E. MahoneyAffiliation 1: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: Department of Cell Systems and Anatomy, UT Health San Antonio, San Antonio, TX, USA
For correspondence: mahoneyr@uthscsa.edu
Bio-protocol author page: a4873
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2401.

Brief version appeared in J Neurosci, Feb 2014
The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous postsynaptic excitatory potentials (mEPSPs) at a mature glutamatergic synapse. Combined with CM9-specific genetic technologies, the CM9 NMJ provides a powerful experimental system to better understand the regulation of neurotransmitter release at a mature synapse.

Isolation of Fucus serratus Gametes and Cultivation of the Zygotes

Featured protocol,  Authors: Amandine Siméon
Amandine SiméonAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: amandine.simeon@sb-roscoff.fr
Bio-protocol author page: a4898
 and Cécile Hervé
Cécile HervéAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: cecile.herve@sb-roscoff.fr
Bio-protocol author page: a4899
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2408.

Brief version appeared in J Exp Bot, Nov 2016
Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials (Kropf et al., 1988; Hervé et al., 2016). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) (Bouget et al., 1998). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus

Featured protocol,  Authors: Eleonora Sendersky
Eleonora SenderskyAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Bio-protocol author page: a4889
Ryan Simkovsky
Ryan SimkovskyAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4890
Susan S. Golden
Susan S. GoldenAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4891
 and Rakefet Schwarz
Rakefet SchwarzAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
For correspondence: Rakefet.Schwarz@biu.ac.il
Bio-protocol author page: a4892
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2406.

Brief version appeared in Sci Rep, Aug 2016
A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue

Featured protocol,  Authors: Jeremy John Mathan
Jeremy John MathanAffiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, the University of Auckland, Private Bag 92019, Auckland, New Zealand
Bio-protocol author page: a4911
Salim Ismail
Salim IsmailAffiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, the University of Auckland, Private Bag 92019, Auckland, New Zealand
Bio-protocol author page: a4912
Jennifer Jane McGhee
Jennifer Jane McGheeAffiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, the University of Auckland, Private Bag 92019, Auckland, New Zealand
Bio-protocol author page: a4913
 and Trevor Sherwin
Trevor SherwinAffiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, the University of Auckland, Private Bag 92019, Auckland, New Zealand
For correspondence: t.sherwin@auckland.ac.nz
Bio-protocol author page: a4914
date: 7/20/2017, 14 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2412.

Brief version appeared in Stem Cell Res Ther, Jun 2016
Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.

Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader

Featured protocol,  Authors: Magdiel Martínez
Magdiel MartínezAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
Bio-protocol author page: a4908
Namyr A. Martínez
Namyr A. MartínezAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
Bio-protocol author page: a4909
 and Walter I. Silva
Walter I. SilvaAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
For correspondence: walter.silva@upr.edu
Bio-protocol author page: a4910
date: 7/20/2017, 19 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2411.

Brief version appeared in J Biol Chem, Jun 2016
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 using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.

Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

Featured protocol,  Authors: Huanhuan Hao
Huanhuan HaoAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4919
Jing Huang
Jing HuangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a2481
Tongtong Liu
Tongtong LiuAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4923
Hui Tang
Hui TangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4921
 and Liping Zhang
Liping ZhangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
For correspondence: zhanglphbu@sohu.com
Bio-protocol author page: a4920
date: 7/20/2017, 7 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2415.

Brief version appeared in Anal Biochem, Sep 2016
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.

Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts

Featured protocol,  Authors: Christopher Hine
Christopher HineAffiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195 USA
For correspondence: hinec@ccf.org
Bio-protocol author page: a4788
 and James R. Mitchell
James R. MitchellAffiliation: Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115 USA
For correspondence: jmitchel@hsph.harvard.edu
Bio-protocol author page: a4789
date: 7/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2382.

Brief version appeared in Cell, Jan 2015
Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H2S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H2S production capacity from biological tissues. H2S released into the head space above a biological sample reacts with lead acetate to form lead sulfide, which is measured on a continuous basis using a plate reader or as an endpoint assay.

Vaginal HSV-2 Infection and Tissue Analysis

Featured protocol,  Authors: Marie Beck Iversen
Marie Beck IversenAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4818
Søren Riis Paludan
Søren Riis PaludanAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4819
 and Christian Kanstrup Holm
Christian Kanstrup HolmAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
For correspondence: holm@biomed.au.dk
Bio-protocol author page: a4820
date: 7/5/2017, 188 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2383.

Brief version appeared in Nat Immunol, Feb 2016
The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even though this is morphologically different from the human vagina, it closely resembles the endocervical epithelium, which is thought to be the primary site of HSV-2 infection in women (Parr et al., 1994; Kaushic et al., 2011). In the protocol presented here, mice are pre-treated with Depo-Provera before intra-vaginal inoculation with HSV-2. The virus replicates in the mucosal epithelium from where it spreads to and replicates in the CNS including the spinal cord, brain stem, cerebrum and cerebellum. Cytokine responses can be detected in vaginal washings using ELISA or in vaginal tissues using qPCR. Further, the recruitment of leukocytes to the vagina can be determined by flow cytometry. The model is suitable for research of both innate and adaptive immunity to HSV-2 infection.

Generation of a Cellular Reporter for Functional BRD4 Inhibition

Featured protocol,  Authors: Sara Sdelci
Sara SdelciAffiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, Vienna, Austria
Bio-protocol author page: a4753
 and Stefan Kubicek
Stefan KubicekAffiliation 1: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, Vienna, Austria
Affiliation 2: Christian Doppler Laboratory for Chemical Epigenetics and Antiinfectives, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
For correspondence: skubicek@cemm.oeaw.ac.at
Bio-protocol author page: a4754
date: 7/5/2017, 177 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2368.

Brief version appeared in Nat Chem Biol, Jul 2016
The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many tumours. Even though BRD4 is more and more studied, its mechanism of action has not been fully described yet. Therefore, we aimed at generating a cellular reporter system to monitor BRD4 inhibition. Such reporter can be potentially used in high throughput chemical and genetic screenings, in order to uncover new possible BRD4 functional pathways. The deeper understanding of the mechanism of action of BRD4 activity will certainly help in developing new therapy strategies for those cancers so called BRD4-dependent.

Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

Featured protocol,  Authors: Anna Fuller
Anna FullerAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4810
Aaron Wall
Aaron WallAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4811
Michael D Crowther
Michael D CrowtherAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4812
Angharad Lloyd
Angharad LloydAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4813
Alexei Zhurov
Alexei ZhurovAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
Bio-protocol author page: a4814
Andrew K. Sewell
Andrew K. SewellAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4815
David K. Cole
David K. ColeAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
For correspondence: ColeDK@cf.ac.uk
Bio-protocol author page: a4816
 and Konrad Beck
Konrad BeckAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
For correspondence: BeckK@cf.ac.uk
Bio-protocol author page: a4799
date: 7/5/2017, 205 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2366.

Brief version appeared in J Clin Invest, Jun 2016
T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis

Featured protocol,  Authors: Imre Gáspár
Imre GáspárAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: imre.gaspar@embl.de
Bio-protocol author page: a4784
 and Anne Ephrussi
Anne EphrussiAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: anne.ephrussi@embl.de
Bio-protocol author page: a4785
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2380.

Brief version appeared in EMBO J, Feb 2017
Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila

Featured protocol,  Author: Dagan Segal
Dagan SegalAffiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
For correspondence: dagansegal@gmail.com
Bio-protocol author page: a4782
date: 7/5/2017, 164 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2377.

Brief version appeared in Dev Cell, Aug 2016
The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large number of fusion events, the structural similarity to vertebrate muscles, and the amenability to the powerful genetic techniques of the Drosophila system make the IFMs an attractive system to study muscle cell fusion. Here we describe methods for live imaging of IFMs, both in intact pupae, and in isolated IFMs ex-vivo. The protocols elaborated upon here were used in the manuscript by (Segal et al., 2016).

Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae

Featured protocol,  Authors: Atit A. Patel
Atit A. PatelAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
Bio-protocol author page: a4794
 and Daniel N. Cox
Daniel N. CoxAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
For correspondence: dcox18@gsu.edu
Bio-protocol author page: a4795
date: 7/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2388.

Brief version appeared in Curr Biol, Dec 2016
To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling in vivo studies of intracellular calcium dynamics in intact Drosophila larvae. We identified Drosophila class III multidendritic (md) sensory neurons as multimodal sensors of innocuous mechanical and noxious cold stimuli and to dissect the mechanistic bases of multimodal sensory processing we developed two independent functional assays. First, we developed an optogenetic dose response assay to assess whether levels of neural activation contributes to the multimodal aspects of cold sensitive sensory neurons. Second, we utilized CaMPARI, a photo-switchable calcium integrator that stably converts fluorescence from green to red in presence of high intracellular calcium and photo-converting light, to assess in vivo functional differences in neural activation levels between innocuous mechanical and noxious cold stimuli. These novel assays enable investigations of behavioral and functional roles of peripheral sensory neurons and multimodal sensory processing in Drosophila larvae.

Tumorigenicity Assay in Nude Mice

Featured protocol,  Authors: Feng Du
Feng DuAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, China
Bio-protocol author page: a4714
Xiaodi Zhao
Xiaodi ZhaoAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, China
For correspondence: leedyzhao@fmmu.edu.cn
Bio-protocol author page: a4715
 and Daiming Fan
Daiming FanAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China
Bio-protocol author page: a4716
date: 7/5/2017, 178 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2364.

Brief version appeared in J Cell Biol, Aug 2015
Tumorigenicity refers to the ability of cultured cells to develop viable tumors in immune-deficient animals. The goal of this protocol is to illustrate tumorigenicity assay by subcutaneous tumor-cell-transplantation in nude mice. Target cells are transplanted to 6-week-old nude mice subcutaneously and the tumor growth is monitored over a period of observation or treatment. When tumor grows to a pre-determined size or by the end of the limited period, the nude mice will be euthanatized and the xenograft will removed for further examination.

A Novel Mouse Skin Graft Model of Vascular Tumors Driven by Akt1

Featured protocol,  Authors: Thuy L. Phung
Thuy L. PhungAffiliation: Department of Pathology, Texas Children’s Hospital and Baylor College of Medicine, Houston, USA
For correspondence: tphung@bcm.edu
Bio-protocol author page: a4800
 and Sriram Ayyaswamy
Sriram AyyaswamyAffiliation: Department of Pathology, Texas Children’s Hospital and Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4801
date: 7/5/2017, 183 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2369.

Brief version appeared in Cancer Res, Jan 2015
To investigate whether endothelial Akt1 activation is sufficient to induce vascular tumor formation in the skin, we have developed a skin graft model in which a skin fragment from transgenic donor mice with inducible and endothelial cell-specific overexpression of activated Akt1 (myrAkt1) is grafted into the skin of wild type recipient mice. The donor skin successfully engrafts after two weeks and, more importantly, vascular tumor develops at the site of transgenic skin graft when myrAkt1 expression is turned on. This skin graft model is a novel approach to investigate the biological impact of a key signal transduction molecule in a temporal, localized and organ-specific manner.

EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells

Featured protocol,  Authors: Yuki Tanaka*
Yuki TanakaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4755
Yasunobu Arima*
Yasunobu ArimaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4756
Kotaro Higuchi
Kotaro HiguchiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4757
Takuto Ohki
Takuto OhkiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4758
Mohamed Elfeky
Mohamed ElfekyAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4759
Mitsutoshi Ota
Mitsutoshi OtaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4760
Daisuke Kamimura
Daisuke KamimuraAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: kamimura@igm.hokudai.ac.jp
Bio-protocol author page: a4761
 and Masaaki Murakami
Masaaki MurakamiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: murakami@igm.hokudai.ac.jp
Bio-protocol author page: a4762
 (*contributed equally to this work) date: 7/5/2017, 203 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2370.

Brief version appeared in Elife, Aug 2015
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system (CNS). It is characterized by focal demyelination and inflammatory responses mediated by myelin-specific autoreactive CD4+ T cells. Using a passive transfer model of EAE in mice, we have demonstrated that regional specific neural signals by sensory-sympathetic communications create gateways for immune cells at specific blood vessels of the CNS, a phenomenon known as the gateway reflex (Arima et al., 2012; Tracey, 2012; Arima et al., 2013; Sabharwal et al., 2014; Arima et al., 2015b). Here we describe protocols for passive transfer model of EAE using freshly isolated (MOG)-specific CD4+ T cells or periodically restimulated MOG-specific CD4+ T cell lines, which are suitable for tracking pathogenic CD4+ T cells in vivo, particularly in the CNS (Ogura et al., 2008; Arima et al., 2012 and 2015b).

Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity

Featured protocol,  Authors: Peter C Petersen
Peter C PetersenAffiliation: Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
Present address: New York University Neuroscience Institute, New York University, New York, New York 10016, USA
Bio-protocol author page: a4786
 and Rune W Berg
Rune W Berg Affiliation: Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
For correspondence: runeb@sund.ku.dk
Bio-protocol author page: a4787
date: 7/5/2017, 229 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2381.

Brief version appeared in Elife, Oct 2016
Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also known as the integrated preparation for electrophysiological investigation. Here, the hind-limb scratch reflex can be induced by mechanical sensory activation, while recording single cells, and the network activity, via intracellular-, extracellular- and electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.

Photothrombotic Induction of Capillary Ischemia in the Mouse Cortex during in vivo Two-Photon Imaging

Featured protocol,  Authors: Robert G. Underly
Robert G. UnderlyAffiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
Bio-protocol author page: a4783
 and Andy Y. Shih
Andy Y. ShihAffiliation 1: Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
Affiliation 2: Center for Biomedical Imaging, Medical University of South Carolina, Charleston, SC, USA
For correspondence: shiha@musc.edu
Bio-protocol author page: a555
date: 7/5/2017, 158 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2378.

Brief version appeared in J Neurosci, Nov 2016
Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al., 2006; Nishimura et al., 2007; Shih et al., 2013). Here we show that small regions of the capillary bed can similarly be occluded to study the ischemic response within the capillary system of the mouse cerebral cortex. The advantage of this approach is that the ischemic zone is restricted to a diameter of approximately 150-250 μm. This permits higher quality two-photon imaging of degenerative processes that would be otherwise difficult to visualize with models of large-scale stroke, due to excessive photon scattering. A consequence of capillary occlusion is leakage of the blood-brain barrier (BBB). Here, through the use of two-photon imaging data sets, we show how to quantify capillary leakage by determining the spatial extent and localization of intravenous dye extravasation.

In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

Featured protocol,  Authors: Dongsheng Jiang
Dongsheng JiangAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
Present address: Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany
For correspondence: dongsheng.jiang@helmholtz-muenchen.de
Bio-protocol author page: a4790
Mona Saffarzadeh
Mona SaffarzadehAffiliation: Department of Biochemistry, School of Medicine, Justus-Liebig-University of Giessen, Giessen, Germany
Present address: Centre for Thrombosis and Haemostasis, University Medical Centre of Mainz, Mainz, Germany
Bio-protocol author page: a4791
 and Karin Scharffetter-Kochanek
Karin Scharffetter-KochanekAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
For correspondence: karin.scharffetter-kochanek@uniklinik-ulm.de
Bio-protocol author page: a4792
date: 7/5/2017, 176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2386.

Brief version appeared in Stem Cells, Sep 2016
In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea

Featured protocol,  Authors: Giovanna Salbitani
Giovanna SalbitaniAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4677
Claudia Bottone
Claudia BottoneAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
Bio-protocol author page: a4765
 and Simona Carfagna
Simona CarfagnaAffiliation: Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy
For correspondence: simcarfa@unina.it
Bio-protocol author page: a4678
date: 7/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2372.

Brief version appeared in Plant Cell Physiol, Sep 2016
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 spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

Featured protocol,  Authors: Yiyuan Niu
Yiyuan NiuAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4803
Yi Ding
Yi DingAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4804
Xiaolong Wang
Xiaolong WangAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4805
 and Yulin Chen
Yulin ChenAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
For correspondence: chenyulin@nwafu.edu.cn
Bio-protocol author page: a4802
date: 7/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2385.

Brief version appeared in Sci Rep, Aug 2016
Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells

Featured protocol,  Authors: Ji Yong Lee
Ji Yong LeeAffiliation: Institute for BioMedical Convergence, Catholic Kwandong University-International St. Mary’s Hospital, Incheon-si, Republic of Korea
Bio-protocol author page: a4768
Seong-Mi Choi
Seong-Mi ChoiAffiliation: Institute for BioMedical Convergence, Catholic Kwandong University-International St. Mary’s Hospital, Incheon-si, Republic of Korea
Bio-protocol author page: a4769
 and Han-Soo Kim
Han-Soo KimAffiliation: Department of Biomedical Sciences, College of Medical Convergence, Catholic Kwandong University, Gangneung-si, Gangwon-do, Republic of Korea
For correspondence: hankim63@gmail.com
Bio-protocol author page: a4770
date: 7/5/2017, 212 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2375.

Brief version appeared in Sci Rep, Sep 2016
Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to distant cells, thereby affecting their gene expression, proliferation, differentiation, and function. Although their physiological functions are not clearly defined, recent studies have shown their therapeutic potential for tissue repair and regeneration. While MVs can be isolated readily from mesenchymal stem cells (MSCs) and other cell types from various sources, the yield of MVs under conventional culture condition in vitro is one of the limiting factors for both the in vivo functional study as well as in vitro molecular analysis. Here, we provide a protocol to increase the yield of microvesicles by preconditioning MSCs with rat brain extract.

Bacterial Survival in Dictyostelium

Featured protocol,  Authors: Regin Rønn*
Regin RønnAffiliation 1: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Affiliation 2: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4771
Xiuli Hao*
Xiuli HaoAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4772
Freja Lüthje
Freja LüthjeAffiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Bio-protocol author page: a4773
Nadezhda A. German
Nadezhda A. GermanAffiliation: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX, USA
Bio-protocol author page: a4774
Xuanji Li
Xuanji LiAffiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Bio-protocol author page: a4775
Fuyi Huang
Fuyi HuangAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4776
Javan Kisaka
Javan KisakaAffiliation: Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA
Present address: Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA
Bio-protocol author page: a4777
David Huffman
David HuffmanAffiliation 1: Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA
Affiliation 2: Department of Plant and Environmental Sciences, University of Copenhagen, MO, USA
Bio-protocol author page: a4778
Hend A. Alwathnani
Hend A. AlwathnaniAffiliation: Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia
Bio-protocol author page: a4779
Yong-Guan Zhu
Yong-Guan ZhuAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4780
 and Christopher Rensing
Christopher RensingAffiliation 1: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Affiliation 2: J. Craig Venter Institute, La Jolla, CA, USA
Affiliation 3: Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture & Forestry University, Fuzhou, China
For correspondence: rensing@iue.ac.cn
Bio-protocol author page: a4781
 (*contributed equally to this work) date: 7/5/2017, 158 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2376.

Brief version appeared in Mol Microbiol, Nov 2016
We performed an assay to test the ability of different E. coli strains to survive inside amoebal cells after ingestion. In the assay we incubated bacteria together with cells of Dictyostelium discoideum for six hours. After co-incubation most of the uningested bacteria were removed by centrifugation and the remaining uningested bacteria were killed by gentamicin. Gentamicin is used because it does not penetrate into eukaryotic cells allowing the ingested bacteria to survive the antibiotic treatment, whereas bacteria outside the amoebal cells are killed.

A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.)

Featured protocol,  Authors: Lee A. Hadwiger
Lee A. HadwigerAffiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA
For correspondence: chitosan@wsu.edu
Bio-protocol author page: a4744
 and Kiwamu Tanaka
Kiwamu TanakaAffiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA
Bio-protocol author page: a4745
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2362.

Brief version appeared in Molecules, Dec 2014
Phytoalexins are antimicrobial substance synthesized in plants upon pathogen infection. Pisatin (Pisum sativum phytoalexin) is the major phytoalexin in pea, while it is also a valuable indicator of plant defense response. Pisatin can be quantitated in various methods from classical organic chemistry to Mass-spectrometry analysis. Here we describe a procedure with high reproducibility and simplicity that can easily handle large numbers of treatments. The method only requires a spectrophotometer as laboratory equipment, does not require any special analytical instruments (e.g., HPLC, mass spectrometers, etc.) to measure the phytoalexin molecule quantitatively, i.e., most scientific laboratories can perform the experiment.

Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth

Featured protocol,  Authors: Natalia V. Andreeva
Natalia V. AndreevaAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Bio-protocol author page: a4766
 and Alexander V. Belyavsky
Alexander V. BelyavskyAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
For correspondence: abelyavs@yahoo.com
Bio-protocol author page: a4767
date: 7/5/2017, 225 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2374.

Brief version appeared in Anal Biochem, Dec 2016
Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins due to their weak non-covalent association with the tissue culture plastic. Here we describe a simple protocol for a controlled fixation of fibronectin, vitronectin and collagen IV layers by formaldehyde, which substantially enhances the stimulation of primary cell proliferation by these extracellular proteins.

Glioma Induction by Intracerebral Retrovirus Injection

Authors: Ravinder K Verma
Ravinder K Verma Affiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Bio-protocol author page: a4878
Fanghui Lu
Fanghui LuAffiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Bio-protocol author page: a4879
 and Qing Richard Lu
Qing Richard LuAffiliation: Division of Experimental Hematology and Cancer Biology, Brain Tumor Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
For correspondence: richard.lu@cchmc.org
Bio-protocol author page: a4880
date: 7/20/2017, 22 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2404.

[Abstract] Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide ...

Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs)

Authors: Wei Xu
Wei XuAffiliation 1: Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Affiliation 2: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
Bio-protocol author page: a4915
 and Chengbiao Wu
Chengbiao WuAffiliation: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
For correspondence: chw049@ucsd.edu
Bio-protocol author page: a4916
date: 7/20/2017, 16 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2413.

[Abstract] The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) is believed to be involved in many disorders of the brain ...

Non-radioactive LATS in vitro Kinase Assay

Authors: Audrey W. Hong
Audrey W. HongAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
Bio-protocol author page: a4827
 and Kun-Liang Guan
Kun-Liang GuanAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
For correspondence: kuguan@ucsd.edu
Bio-protocol author page: a868
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2391.

[Abstract] This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate....

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

[Abstract] Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance ...

Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

[Abstract] Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly ...

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

[Abstract] Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as ...

Plasmodium Sporozoite Motility on Flat Substrates

Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

[Abstract] Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro. ...

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster

Authors: Benjamin A. Eaton
Benjamin A. EatonAffiliation 1: Department of Cellular and Integrative Physiology, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Bio-protocol author page: a4872
 and Rebekah E. Mahoney
Rebekah E. MahoneyAffiliation 1: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: Department of Cell Systems and Anatomy, UT Health San Antonio, San Antonio, TX, USA
For correspondence: mahoneyr@uthscsa.edu
Bio-protocol author page: a4873
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2401.

[Abstract] The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous ...

Isolation of Fucus serratus Gametes and Cultivation of the Zygotes

Authors: Amandine Siméon
Amandine SiméonAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: amandine.simeon@sb-roscoff.fr
Bio-protocol author page: a4898
 and Cécile Hervé
Cécile HervéAffiliation: CNRS–UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, Roscoff, France
For correspondence: cecile.herve@sb-roscoff.fr
Bio-protocol author page: a4899
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2408.

[Abstract] Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually ...

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus

Authors: Eleonora Sendersky
Eleonora SenderskyAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Bio-protocol author page: a4889
Ryan Simkovsky
Ryan SimkovskyAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4890
Susan S. Golden
Susan S. GoldenAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4891
 and Rakefet Schwarz
Rakefet SchwarzAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
For correspondence: Rakefet.Schwarz@biu.ac.il
Bio-protocol author page: a4892
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2406.

[Abstract] A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile ...
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Scratch Wound Healing Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 57761 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.100.

[Abstract] 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 ...

In vitro Culture of Human PBMCs

Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
date: 2/5/2013, 45752 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.322.

[Abstract] 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 ...

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 2/20/2012, 44518 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.99.

[Abstract] 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, ...

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 44030 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] 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 ...

Clonogenic Assay

Author: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
date: 5/20/2012, 40781 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187.

[Abstract] 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 ...

[Bio101] Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)

Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
date: 4/5/2012, 40707 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.198.

[Abstract] Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU ...

Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2012, 36362 views, 15 Q&A
DOI: https://doi.org/10.21769/BioProtoc.68.

[Abstract] 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 ...

In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

Authors: Josephine MY Ko
Josephine MY KoAffiliation: Clinical Oncology Department, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a100
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
date: 9/20/2012, 34088 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.260.

[Abstract] Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to ...

Soft–Agar colony Formation Assay

Author: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
date: 7/5/2012, 32516 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.220.

[Abstract] Any anchorage–independent growth of tumor cells is estimated by a soft–agar colony formation assay. This protocol provides a general workflow for establishing a soft-agar colony formation assay....

[Bio101] Cell Adhesion Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 30364 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.98.

[Abstract] Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization ...
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