McMaster University

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15 articles published in JoVE

Bioengineering

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning Daryl Sivakumaran¹, Emilia Bakaic¹, Scott B. Campbell¹, Fei Xu¹, Eva Mueller¹, Todd Hoare¹ ¹Department of Chemical Engineering, McMaster University Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.
Genetics

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott^1,2, Sali M.K. Farhan³, Mahdi Ghani⁴, Christine Sato⁴, Eric Liang⁵, Ming Zhang⁴, Adam D. McIntyre¹, Henian Cao¹, Lemuel Racacho^6,7, John F. Robinson¹, Michael J. Strong^1,8, Mario Masellis^9,10, Dennis E. Bulman^6,7, Ekaterina Rogaeva⁴, Anthony Lang^10,11, Carmela Tartaglia^4,10, Elizabeth Finger^12,13, Lorne Zinman⁹, John Turnbull¹⁴, Morris Freedman^10,15, Rick Swartz⁹, Sandra E. Black^9,16, Robert A. Hegele^1,2 ¹Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, ²Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, ³Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, ⁴Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, ⁵ Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.
Chemistry

Preparation and Evaluation of ^99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry Holly A. Bilton*¹, Zainab Ahmad*¹, Nancy Janzen¹, Shannon Czorny¹, John F. Valliant¹ ¹Department of Chemistry and Chemical Biology, McMaster University Here, we describe a protocol for radiolabeling and in vivo testing of tridentate ^99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.
Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae Alicja Puchta¹, Chris P. Verschoor¹, Tanja Thurn¹, Dawn M. E. Bowdish¹ ¹Department of Pathology and Molecule Medicine, McMaster University Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.
Medicine

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice Jane A. E. Batt¹, James Ralph Bain² ¹Keenan Research Centre of the LiKaShing Knowledge Institute, St Michaels Hospital, ²Department of Surgery, McMaster University The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.
Chemistry

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays Blake-Joseph Helka¹, John D. Brennan¹ ¹Department of Chemistry and Chemical Biology, McMaster University A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.
Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion Justin Tong¹, Pouya Rezai², Sangeena Salam¹, P. Ravi Selvaganapathy², Bhagwati P. Gupta¹ ¹Department of Biology, McMaster University, ²Department of Mechanical Engineering, McMaster University A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (“electrotaxis”) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.
Biology

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes Yu Seon Chung¹, Alba Guarné¹ ¹Department of Biochemistry and Biomedical Sciences, McMaster University Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.
Medicine

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting Chitra Venugopal¹, Nicole M. McFarlane¹, Sara Nolte¹, Branavan Manoranjan¹, Sheila K. Singh¹ ¹Stem Cell and Cancer Research Institute, McMaster University The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
Immunology and Infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation Rahul Kushwah¹, Jim Hu² ¹Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ²Physiology and Experimental Medicine Research Program, Hospital for Sick Children, University of Toronto We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.
Immunology and Infection

Detection of Bacteria Using Fluorogenic DNAzymes Sergio D. Aguirre¹, M. Monsur Ali¹, Pushpinder Kanda¹, Yingfu Li^1,2 ¹Department of Biochemistry and Biomedical Sciences, McMaster University, ²Department of Chemistry and Chemical Biology, McMaster University We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).
Biology

Investigating Intestinal Inflammation in DSS-induced Model of IBD Janice J. Kim¹, Md. Sharif Shajib¹, Marcus M. Manocha¹, Waliul I. Khan¹ ¹Farncombe Family Digestive Health Research Institute, Department of Pathology and Molecular Medicine, McMaster University Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.
Biology

Primary Human Bronchial Epithelial Cells Grown from Explants Asma Yaghi¹, Aisha Zaman¹, Myrna Dolovich¹ ¹Medicine, Faculty of Health Sciences, McMaster University Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.
Biology

A Tactile Automated Passive-Finger Stimulator (TAPS) Daniel Goldreich^1,2, Michael Wong², Ryan M. Peters², Ingrid M. Kanics¹ ¹Department of Occupational Therapy, Duquesne University, ²Department of Psychology, Neuroscience and Behaviour, McMaster University We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Biology

Culture of myeloid dendritic cells from bone marrow precursors Jeanette Boudreau^1,2, Sandeep Koshy^2,3, Derek Cummings², Yonghong Wan² ¹Medical Sciences Program, McMaster University, ²Centre for Gene Therapeutics, McMaster University, ³Department of Chemical Engineering, University of Waterloo This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

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