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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.

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.

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Featured protocol,  Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

Brief version appeared in PLoS Pathog, Feb 2017
Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides

Featured protocol,  Authors: Marina Shenkman
Marina ShenkmanAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Bio-protocol author page: a4830
Navit Ogen-Shtern
Navit Ogen-ShternAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Present address: the Skin Research Institute, The Dead Sea and Arava Science Center, Masada, Israel
Bio-protocol author page: a4831
 and Gerardo Z. Lederkremer
Gerardo Z. LederkremerAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
For correspondence: Gerardo@post.tau.ac.il
Bio-protocol author page: a4832
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2393.

Brief version appeared in J Mol Biol, Aug 2016
Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

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.

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.

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen

Featured protocol,  Authors: Brent Veerman
Brent VeermanAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a4752
 and Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: ritu.chakravarti@utoledo.edu
Bio-protocol author page: a3737
date: 7/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2367.

Brief version appeared in Arthritis Rheumatol, Jul 2015
Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering with large vessel vasculitis (LVV), an autoimmune disease (Chakravarti et al., 2015). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient’s sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% (Chakravarti et al., 2015). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.

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).

Social Observation Task in a Linear Maze for Rats

Featured protocol,  Authors: Xiang Mou
Xiang MouAffiliation 1: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA
Affiliation 2: Memory and Brain Research Center, Baylor College of Medicine, Houston, USA
For correspondence: xmou@bcm.edu
Bio-protocol author page: a4742
 and Daoyun Ji
Daoyun JiAffiliation 1: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, USA
Affiliation 2: Department of Neuroscience, Baylor College of Medicine, Houston, USA
Affiliation 3: Memory and Brain Research Center, Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4743
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2361.

Brief version appeared in Elife, Oct 2016
Animals often learn through observing their conspecifics. However, the mechanisms of them obtaining useful knowledge during observation are beginning to be understood. This protocol describes a novel social observation task to test the ‘local enhancement theory’, which proposes that presence of social subjects in an environment facilitates one’s understanding of the environments. By combining behavior test and in vivo electrophysiological recording, we found that social observation can facilitate the observer’s spatial representation of an unexplored environment. The task protocol was published in Mou and Ji, 2016.

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.

Delayed-matching-to-place Task in a Day Maze to Measure Spatial Working Memory in Mice

Featured protocol,  Authors: Xi Feng
Xi FengAffiliation 1: Brain and Spinal Injury Center, University of California San Francisco, San Francisco, California, USA
Affiliation 2: Departments of Physical Therapy and Rehabilitation Science, University of California San Francisco, San Francisco, California, USA
For correspondence: xi.feng@ucsf.edu
Bio-protocol author page: a3096
Karen Krukowski
Karen KrukowskiAffiliation 1: Brain and Spinal Injury Center, University of California San Francisco, San Francisco, California, USA
Affiliation 2: Departments of Physical Therapy and Rehabilitation Science, University of California San Francisco, San Francisco, California, USA
Bio-protocol author page: a4796
Timothy Jopson
Timothy JopsonAffiliation 1: Brain and Spinal Injury Center, University of California San Francisco, San Francisco, California, USA
Affiliation 2: Departments of Physical Therapy and Rehabilitation Science, University of California San Francisco, San Francisco, California, USA
Bio-protocol author page: a4797
 and Susanna Rosi
Susanna RosiAffiliation 1: Brain and Spinal Injury Center, University of California San Francisco, San Francisco, California, USA
Affiliation 2: Departments of Physical Therapy and Rehabilitation Science, University of California San Francisco, San Francisco, California, USA
Affiliation 3: Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA
Affiliation 4: Weill Institute for Neuroscience, University of California San Francisco, San Francisco, California, USA
Affiliation 5: Kavli Institute of Fundamental Neuroscience, University of California San Francisco, San Francisco, California, USA
For correspondence: susanna.rosi@ucsf.edu
Bio-protocol author page: a4798
date: 7/5/2017, 157 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2389.

Brief version appeared in J Neuroinflammation, Aug 2016
The delayed-matching-to-place (DMP) dry maze test is a variant of DMP water maze (Steele and Morris, 1999; Faizi et al., 2012) which measures spatial working/episodic-like learning and memory that depends on both hippocampal and cortical functions (Wang and Morris, 2010; Euston et al., 2012). Using this test we can detect normal aging related spatial working memory decline, as well as trauma induced working memory deficits. Furthermore, we recently reported that fractionated whole brain irradiation does not affect working memory in mice (Feng et al., 2016). Here we describe the experimental setup and procedures of this behavioral test.

Active Cdk5 Immunoprecipitation and Kinase Assay

Featured protocol,  Authors: Andrew N. Bankston
Andrew N. BankstonAffiliation 1: Department of Pharmacology, Emory University, Atlanta, GA
Affiliation 2: Department of Neurological Surgery, University of Louisville, Louisville, KY
Bio-protocol author page: a4746
Li Ku
Li KuAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
Bio-protocol author page: a4747
 and Yue Feng
Yue FengAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
For correspondence: yfeng@emory.edu
Bio-protocol author page: a4748
date: 7/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2363.

Brief version appeared in J Biol Chem, Jun 2013
Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal amounts of total protein between experimental groups. Washes are then performed to remove extraneous proteins and equilibrate the Cdk5-activator complexes in the kinase buffer. Cdk5 is then incubated with histone H1, a well-established in vitro target of Cdk5, and [γ-32P]ATP. Reactions are resolved by SDS-PAGE and transferred to membranes for visualization of H1 phosphorylation and immunoblot of immunoprecipitated Cdk5 levels. We have used this assay to establish p39 as the primary activator for Cdk5 in the oligodendroglial lineage. However, this assay is amenable to other cell lineages or tissues with appropriate adjustments made to lysis conditions.

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.

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 ...

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 ...

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, ...

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 ...

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan ...

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides

Authors: Marina Shenkman
Marina ShenkmanAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Bio-protocol author page: a4830
Navit Ogen-Shtern
Navit Ogen-ShternAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Present address: the Skin Research Institute, The Dead Sea and Arava Science Center, Masada, Israel
Bio-protocol author page: a4831
 and Gerardo Z. Lederkremer
Gerardo Z. LederkremerAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
For correspondence: Gerardo@post.tau.ac.il
Bio-protocol author page: a4832
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2393.

[Abstract] Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation ...

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue

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.

[Abstract] 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 ...

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

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.

[Abstract] 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 ...

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

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.

[Abstract] 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 ...
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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] Glucose Tolerance Test in Mice

Author: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 10/5/2011, 32159 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.159.

[Abstract] Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from blood. Specifically, uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e, longer time to clear given amount of glucose) indicates problems ...
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