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Biology of Breathing (BoB) has established and operates five core research facilities equipped with multiple research instruments. The use of these facilities by local and external investigators to complete collaborative research projects with BoB investigators is encouraged. To obtain information about equipment and access, and to plan research projects, trainees and investigators should e-mail the Children’s Hospital Research Institute of Manitoba Facility & Operations Manager, Nichola Wigle (email@example.com) and the Program Director(s) and/or the Senior Technologist/Manager(s) listed below.
Located in the Children’s Hospital Research Institute of Manitoba on the 6th floor of the John Buhler Research Centre, the facility includes three instruments:
1. Two-colour Olympus FluoView Scanning Confocal Microscope.
The system includes an inverted Olympus LX70 microscope equipped with krypton and argon lasers that generate 3 different excitation lines, and has filter cubes for detection of green, red and far red emission. The instrument can also be used to obtain difference interference contrast (DIC) images cells and tissue. Specialized software is used for 2-dimentional and 3-dimentional image capture and analysis.
2. Inverted epi-fluorescence Microscope with Filter Wheel for Real Time Ratiometric Imaging in Live Cells.
The system includes an inverted Olympus IX70 microscope with filter cubes to capture blue, green, red, and far-red fluorescence. It also has a Lambda filter wheel configured for studies with fura-2 to measure time lapse changes in intracellular free Ca2+ in living cells. The system has recently been upgraded with the addition of a new high resolution CCD camera and Nikon NIS software.
3. Research Laser Scanning Cytometer (LSC).
We operate a CompuCyte iCys LSC equipped with three lasers and that can capture blue, green, orange-red, and far red fluorescence. It has high resolution light scatter detectors for capturing shaded relief (DIC-like) images, and the detectors can be tuned to measure laser light-loss (absorbance), thus allowing analysis of cell and tissue samples prepared with standard chromogenic dyes. The LSC has a micro-step motorized stage that scans cells or tissue samples and captures light intensity and positional data based on operator-specified step size with flexibility for resolution from sub-cellular organelles to the whole tissue level. Sophisticated software collates fluorescence emission or laser light absorbance for each event with positional data thus offering the unique ability to generate a image gallery of cells that correlate to specific gated events (eg. apoptosis). The instrument is routinely used for analysis of cell cycle and apoptosis, mitochondrial membrane potential, intracellular ROS, and coincident marker protein expression in unique cell subpopulations; we encourage investigators to discuss their specific needs so we can develop new protocols.
Director: Dr. J. Elliot Scott (firstname.lastname@example.org)
This facility is located Dr. Scott s lab in the Department of Oral Biology (Rm. 310 Dentistry Building). It is essential for analyzing lung surfactant expression and quality from in vitro systems (cultured Type I alveolar epithelial cells) and in vivo derived samples (lung lavage samples form animal models and human subjects). The facility includes:
1. Captive Bubble Surfactometer
This sophisticated computer-controlled instrument captures pictures of bubbles and analyzes the dynamic properties of surfactant samples to provide a direct measure of their quality.
2. Capillary Surfactometer
This instrument uses capillary tubes to simulate small airways and measures the ability of very small samples of surfactant (~0.5Î¼L) to maintain patency, thereby providing an index of surfactant function. The small volumes that can be analyzed offers unique opportunities for translational research, for example the instrument is being used to analyze breathe condensate samples from clinical research trials with children in collaboration with Dr Pasterkamp.
3. Surfactant Lipid and Protein Analysis Suite
The facility is equipped to isolate, quantify and characterize surfactant lipids (eg. DPPC) and proteins using thin layer chromatography and protein blotting, respectively.
Co-Directors: Dr. Ganesh Srinivasan (email@example.com) and Dr. Andrew Halayko (firstname.lastname@example.org)
Senior Technologist/Manager: Ms. Sujata Basu (email@example.com)
To support pre-clinical and translational research this facility has been established for in vivo measurement of respiratory mechanics in mouse models of lung disease, and for ex vivo measurement of constriction and relaxation of airways and blood vessels using micro-dissected isolated preparations or precision cut thin lung slices. We can also provide support for ancillary assessment of cell and molecular markers of inflammation, assessment of lung structure, and development of murine models of disease (eg. chronic asthma). The facility includes:
1. flexiVENT Small Animal Ventilator
This computer-controlled state-of-the-art equipment manufactured by Scireq Inc is located in the Children\’s Hospital Research Institute of Manitoba (Rm 522, John Buhler Research Centre) and offers capacity for invasive assessment of lung function in individual mice. The ventilator includes an in-line nebulizer that can be used to deliver aerosolized research compounds and thereafter measure their affects on respirator mechanics. The instrument is used routinely to measure baseline lung function and the changes that occur in response to inhalation of increasing concentrations of the bronchial spasmogen, methacholine (MCh). We employ low frequency forced oscillation maneuvers in anesthetized mice to derive the sensitivity and reactivity to inhaled MCh for airway resistance (Raw), tissue resistance (G), lung stiffness (H), and we complement these data by capturing static pressure-volume curves.
2. Whole Body Plethysmograph
This computer-controlled apparatus manufactured by Buxco Inc is located in the Children’s Hospital Research Institute of Manitoba (Rm 522, John Buhler Research Centre) and offers capacity for non-invasive assessment of breathing in up to 4 mice simultaneously. The system offers the advantage of being able to make repeated measurements of breathing in mice over several weeks, however it does not provide information about specific parameters of respirator mechanics, rather one can make an assessment of the work of breathing. Like our invasive ventilator, this system includes an in-line nebulizer that can be used to deliver aerosolized research compounds and measure their affects on breathing.
3. Precision Lung Slice Facility
This apparatus is located in the Children’s Hospital Research Institute of Manitoba (Long-term Microscopy Lab in Rm 641, John Buhler Research Centre) and enables video-microscopy analysis of airway and blood vessel constriction and relaxation in thin slices of lungs from animal models or from human specimens. Live lungs are inflated with agarose, cooled and then sectioned using a vibratome to produce 100-200Î¼m thick lung slices that are kept viable for up to 3 days in a culture incubator. Individual slices are mounted in a custom microscope-mounted chamber with constant perfusion and capacity for timed delivery of pharmacological compounds. The microscope is equipped with a CCD camera controlled by Nikon NIS software and captures time lapse images for subsequent quantitative analysis of blood vessel or bronchial constriction, which is essential in testing of new compounds for disease treatment, and can be used to corroborate finding from in vivo lung function assessment.
4. Wire Myograph to Measure Isometric Force
Senior Technologist/Manager: Ms. Nora Nolette (firstname.lastname@example.org)
This sensitive system manufactured by Danish Myo Technology (Atlanta, GA) is located in the Children’s Hospital Research Institute of Manitoba (Rm 641, John Buhler Research Centre) and allows measurement of isometric force generated by micro-dissected preparations of blood vessels or airways. We operate two 4-bath 610M Multi Wire Myograph Systems that can be used to study ring preparations with diameters from 60 µm (resistance arteries) to 10mm (tracheal and bronchial rings). The facility allows assessment of isolated blood vessel and airway function, which is essential in early testing of new compounds for disease treatment, and can be used to complete pharmacological studies to corroborate finding from in vivo lung function assessment.
Senior Technologist/Manager: Ms. Karen Detillieux (email@example.com)
Our facility and core staff can enable investigators to incorporate sophisticated molecular biology techniques into their research programs, and test whether specific gene products may be suitable targets for developing new drugs to treat pediatric lung disease. The facility includes equipment and technical expertise for studies using techniques such as qRT-PC, luciferase reporter assays, or the transfection of cells with plasmid constructs or siRNA using an Amaxa Nucleofector. Our technologists offer support for PCR primer design and for using adenoviral, retroviral and lentiviral vectors to transduce cells to manipulate gene expression and generate stable cell lines. Core technologists serve a managerial role in the Viral Culture Lab at the Children’s Hospital Research Institute of Manitoba (Rm 615, John Buhler Research Centre) and a close working relationship with the Manitoba Centre for Proteomics and Systems Biology that has acquired The RNA Consortium and Cold Spring Harbor Laboratories human and mouse lentiviral RNAi libraries through Open Biosystems ®. These libraries provide unique capability to selectively silence any gene in human and mouse cells and identify their role in cell function.
Director: Dr. Shyamala Dakshinamurti (firstname.lastname@example.org)
This facility has been established in the animal holdings facility at the St. Boniface Research Centre. The lab provides capacity to assess cardio-pulmonary physiology in large animal models, such as the piglets used in Dr. Dakshinamurti s studies of persistent pulmonary hypertension of the newborn. Investigators are encouraged to contact Dr. Dakshinamurti to discuss the potential utility of this facility for their own work.