The ultimate goal of the MMRL’s Molecular Genetics Core is to identify the factors that are responsible for diseases. This knowledge will facilitate the development of gene-specific therapies and cures for arrhythmias and identify individuals at risk for sudden cardiac deaths.
As researchers discover the role genes play in disease, there will be more genetic tests available to help doctors make diagnoses and pinpoint the cause of the disease. For example, heart disease can be caused either by a mutation in certain genes, or by environmental factors such as diet or exercise to name a few.
Physicians can easily diagnose a person with heart disease once they present symptoms. However, physicians can not easily identify the cause of the heart disease is in each person. Thus, most patients receive the same treatment regardless of underlying cause of the disease.
In the future, a panel of genetic tests for heart disease might reveal the specific genetic factors that are involved in a given person. People with a specific mutation may be able to receive treatment that is directed to that mutation, thereby treating the cause of the disease, rather than just the symptoms.
The ultimate goal of the MMRI’s Molecular Genetics Core is to identify the factors that are responsible for these diseases. This knowledge will facilitate the development of gene-specific therapies and cures for arrhythmias and identify individuals at risk for sudden cardiac deaths.
With the addition of the Molecular Biology and Molecular Genetics cores, MMRI is now integrally involved in both basic and clinical research, and is among the relatively few institutions worldwide with a consistent and concerted focus on bridging basic and clinical science. With an eye toward designing specific treatments.
The Advanced Imaging Core at MMRI was developed to facilitate the non-invasive analysis of preclinical models of disease. The imaging suite is outfitted with state-of-the-art equipment for small animal in vivo imaging using fluorescence, x-ray computed tomography and ultrasound.
Our facility includes:
- Perkin Elmer IVIS Spectrum – 2D and 3D optical imaging
- Perkin Elmer Quantum GX microCT – x-ray computed tomography imaging
- Visual Sonics Vevo 3100 Ultrasound – high frequency ultrasound imaging
2D and 3D optical imaging. An optimized set of high efficiency filters and spectral un-mixing algorithms lets you take full advantage of bioluminescent and fluorescent reporters across the blue to near infrared wavelength region. It also offers single-view 3D tomography for both fluorescent and bioluminescent reporters that can be analyzed in an anatomical context using our Digital Mouse Atlas or registered with our multimodality module to other tomographic technologies such as MR, CT or PET.
For advanced fluorescence pre-clinical imaging, the Perkin Elmer IVIS Spectrum has the capability to use either trans-illumination (from the bottom) or epi-illumination (from the top) to illuminate in vivo fluorescent sources. 3D diffuse fluorescence tomography can be performed to determine source localization and concentration using the combination of structured light and trans illumination fluorescent images. The instrument is equipped with 10 narrow band excitation filters (30nm bandwidth) and 18 narrow band emission filters (20nm bandwidth) that assist in significantly reducing autofluorescence by the spectral scanning of filters and the use of spectral unmixing algorithms. In addition, the spectral unmixing tools allow the researcher to separate signals from multiple fluorescent reporters within the same animal. The instrument possesses 23 cm field of view capable of imaging 5 mice simultaneously in reflectance mode, while maintaining a high resolution (to 20 microns) with 3.9 cm field of view. When used in conjunction with the Quantum µCT, the software can automatically co-register images, yielding functional and anatomical localization of fluorophores.
X-ray computed tomographic imaging. The Perkin Elmer Quantum GX µCT multispecies imaging system provides high-resolution images at an X-ray dose low enough to enable true longitudinal imaging capability. With scan times as low as 8 seconds, the Quantum GX supports a workflow of up to 30 subjects per hour, with acquisition, reconstruction, and 3D visualization in under one minute. In high-resolution mode, the Quantum is capable of producing images with a 4.5 micrometer voxel size. Additionally, the Quantum features two-phase respiratory and cardiac gating allowing for detailed cardiac imaging.
Ultrasound imaging. The VisualSonics Vevo 3100 ultrasound imaging system utilizes a powerful combination of high frame rates and advanced image processing to reduce speckle noise and artifacts while preserving and enhancing critical information for small animal in vivo studies.
The Histopathology Core at MMRI provides a range of histological services, including tissue fixation and processing, paraffin and cryosectioning, common and advanced histological stains, as well as immunohistochemistry and fluorescence staining.
The core facility is equipped with state-of-the-art equipment to provide highest quality specimens for data collection and analysis.
Our facility includes:
- Leica CM1950 cryostat – for fresh frozen and fixed frozen tissue sectioning
- Leica RM2125 RTS manual microtome – for paraffin embedded tissue sectioning
- Keyence BZ-X800 – an “all-in-one” inverted microscope – fluorescence, brightfield, and phase contrast images
- Nikon Ni-E Research Microscope System – upright microscope – fluorescence, brightfield, and polarized light
The Flow Cytometry Core (FCC) at MMRI provides instrumentation and expertise in a broad range of basic and medical science disciplines. Samples are prepared by individual investigators, who then deliver samples to the core for flow cytometric analysis or cell sorting. The core has one sorter (BD FACS Aria Fusion) and one analytical cytometer (BD FACS Symphony A3).
The MMRI Flow Cytometry Core Facility offers the following instruments and capabilities:
BD FACS Symphony A3
Sample Preparation: The minimum recommended volume is 500 µl at a concentration of 1×106 cells per ml and cell suspensions must be placed in polystyrene 12x75mm test tubes after filtering them through 45µm filter.
The BD FACS Aria Fusion is capable of high speed and single cell sorting on up to thirteen parameters (forward & side scatter and 11 simultaneous fluorescence colors). It is equipped with four lasers: 405nm, 488nm, 561nm, and 640nm. The BD FACSAria Fusion has the capability of four-way sorting into tubes or single cell sorting directly into plates or slides
This machine has 4 different nozzles: 70 µm, 85 µm, 100 µm and 130 µm in diameter. It is strongly advised to choose a nozzle size that is 3-5 times the diameter of the cells
Sort Sample Requirements:
The outcome of the cell sorting process depends on sample preparation. Most importantly, it is critical to minimize clumps in the sample to be sorted. Clumping is usually caused by inadequate dissociation of cells and/or cell death and release of DNA from dead cells. For these reasons, we recommend the use of a proper sort buffer containing DNAse, and passing the sample through a hypodermic needle prior to sorting
Recommended sort buffer composition:
- 1x PBS or 1x HBSS (Ca/Mg++ free)
- 2mM EDTA
- 25mM HEPES pH 7.0
- 1-5% FCS/FBS (Heat-Inactivated)
- 10units/mL DNase II
- 0.2um filter sterilize
Ideally, cell concentration should not to exceed 106 cells/mL. Cells are to be filtered through 45 uM mesh just prior to sorting (after final wash or staining step). It is also recommended that you bring your samples inside a syringe fitted with a 25 g needle, especially if sample volumes exceed 500 µl; passing samples through the needle prior to sorting will minimize clumping.
Phenol red is not recommended in cell suspension media
Controls for experiments:
An unstained control and a single positive control for each fluorophore used is required
Controls should be brought in a volume of 0.5-1mL and preferably contain approximately 106 cells
Collection Tube Requirements:
It is recommended to collect sorted cells into 12×75 mm GLASS tubes pre- coated with serum/media. Pre-coating collection tubes with serum/media will increase post-sort viability and recovery, while glass minimizes static deflection of charged sort droplets. Sorting into polystyrene and polypropylene tubes may reduce recovery as cells have a tendency to stick to plastic, and these plastics can also build up a static charge All collection tubes should also be filled approximately ¼ with desired buffer or media. It is also recommended to add antibiotics (1x PSN and/or 50ug/ml gentamicin) are added to culture media for samples sorted for cell culture, especially primary cell culture, to prevent contamination.