Technologies
Contact
1020 Locust Street
Jefferson Alumni Hall, Suite 527
Philadelphia, PA 19107
At MitoCare, we test our ideas by implementing cutting-edge imaging, state-of-the-art mitochondrial phenomics, and relevant in vivo disease models.
Phenomics
Investigators at MitoCare study mitochondria function in whole animals, ex vivo and in vitro.
Seahorse Extracellular Flux Analyzer (XFe24)
Measures O2 consumption rate (OCR) and extracellular acidification rate (ECAR) of adherent cells, in a 24-well format. Recent modeling and computational advances that correct for O2 fluxes unrelated to cellular O2 reduction have resulted in a further increase in sensitivity such that many human primary cells that have low O2 consumption rates can now be reliably studied. Recent advances in experimental design have supplied protocols that allow the ECAR signal to be specific for glycolytic flux. The instrument has been widely endorsed by the bioenergetics community. No other method for adherent cells approaches the sensitivity of the Seahorse instrument.
TYPE of Samples
- Adherent cells
- Isolated mitochondria
- Zebrafish embryos
- Some tissue preps (need for substantial optimization should be expected)
Specifications
- Consumables: Custom cell culture plate and sensor cartridge, only available from Agilent
- Up to 4 injections during an experiment.
- Experimental temperature range: 16 – 42°C
Additional Details
Some information, including details about the technology and some protocols (Agilent): https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-analyzers
Consumables (Agilent):
https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-sensor-cartridges-cell-culture-microplates
Instrument availability:
One instrument is available for use as part of the MitoCare Shared Resources service center.
Oroboros Oxygraph 2K
Measures O2 consumption rate of samples in suspension. Typical samples are isolated mitochondria and cells. The instrument has a two-sample capacity. Its high degree of sensitivity makes it state-of-the-art. The instrument can also be adapted for parallel determinations of O2 consumption and mitochondrial inner membrane potential using a TPMP+ electrode.
OXYMax Indirect Calorimetry System
Measures whole-body O2 consumption and CO2 production. Equipment is housed in a light-cycle controlled dedicated room, with monitoring of room humidity and temperature.
Exercise & Behavior
Rodent treadmill “Exer 3/6” (Columbus Instr.)
Software-controlled for speed, acceleration, and incline. The software also records the distance traveled. Equipment is housed in a dedicated room.
Metabolic modular treadmill for mice (Columbus Instr.)
A one-lane treadmill with the same features as the ‘Rodent Treadmill’, but which can be enclosed with air-tight seals for measuring oxygen consumption during animal exercise through coupling with the OXYMAX indirect Calorimetry System.
Muscle force measurements
- Standalone ASI contractile force measurement equipment for recording skeletal muscle force in vivo or in isolated skeletal muscle during electrical stimulation.
- Microscope adaptable, ASI force transducer and electrical stimulation unit to record force during isometric contraction of skeletal muscle fibers/cardiac trabeculae simultaneously with microscopic observation.
Rotarod (Maze Engineers)
Widely used behavioral task to evaluate motor performance. The instrument can be customized for speed, acceleration, and is controlled using the Conductor Software.
Murine Echocardiography
Multiwell Reader
BIOTEK Synergy neo2
High-end plate reader that measures fluorescence, absorbance, and luminescence. The instrument is designed with separate optics for each of these capabilities; thus each capability has been optimized. The instrument also allows dual-wavelength excitation, and time-resolved fluorescence, and is equipped with an injection system. The instrument can also be programmed to read from a custom Seahorse microplate.
Proteomics
Mitochondrial Mass Spectrometry
Several investigators at MitoCare use mass spectrometry approaches to interrogate the protein composition of different mitochondrial compartments: from mitochondrial DNA nucleoids to the constituents of the mitochondrial-endoplasmic reticulum contact sites. Proteins are purified from different biological samples, in their native state or after isotope tagging (SILAC) or proximity biotinylation.
Gene Profiling
Mitochondrial DNA Expression
An indirect reading of the metabolic capacity of a cell or a tissue is provided by the total amount of mitochondrial DNA (mtDNA) present at any given time, its transcription rate, and the proportion of mutated versus wild-type molecules. We developed and optimized a wide range of assays to evaluate mtDNA copy number (in human and mouse samples) and mutation (i.e. deletion) using real-time quantitative PCR coupled to relative or absolute quantification strategies. Similarly, specific assays are available for mitochondrial gene transcription.
Imaging
MitoCare eyes observe the mitochondria details of a wide range of bio-specimens, with a resolution that can reach 3 nm in 3-axis.
3D imaging capabilities of common TEM is limited to a section thickness of <200 nm, to allow transmission of electrons at high tilt-angles; but >50 nm, which is the thinnest one can cut using diamond knife in an ultramicrotome. The Thermo-Fisher Helios 5 FIB-SEM in MitoCare combines block surface imaging via ultra-high resolution scanning electron microscopy, which is not limited by sample thickness, with serial focused ion beam-mediated milling of the surface with nanometer precision. This way, it can generate tomographic image stacks across several (up to ~20) microns, at high (down to 3-5 nm isotropic voxels) resolution, allowing 3D reconstruction of large cellular subvolumes, to study the membrane details of whole organelles, their network interactions or synaptic cell-cell interactions.
Using electron optics in lieu of light allows low nanometer to subnanometer resolution insights to the ultrastructure of a fixed specimen derived from tissue, cells, or subcellular preparations that have been processed for enhanced electron scattering by the structures of interest and for resisting high vacuum. 2D insights are delivered by a Thermo/FEI Tecnai 12 digital transmission electron microscope (TEM), featuring 120 kV maximum electron beam acceleration voltage and a bottom-mounted AMT BioSprint 12 megapixel High Definition CCD camera. We offer TEM imaging, sample processing (e.g. positive staining, resin-embedding of cells/tissue, negative staining of vesicles or purified proteins), and sectioning services as well as consultations in ultrastructure-directed experimental design and analysis.
Vutara 352 (Bruker) For single-molecule localization (StORM/PALM; can achieve resolution of ~50nm in three dimensions), and for TIRF imaging and single-molecule tracking.
It is equipped with 405, 488, 561, 639 and 750 nm lasers and performs detection via Quad-Field projection onto a sCMOS camera that allows simultaneous imaging of two colors in two focal planes or up to four colors in a single-plane. Includes a motorized TIRF module and temperature-controlled incubation for live cell imaging. Includes software for analysis of single-molecule localization and quantitative analysis of STORM/PALM experiments.
Zeiss LSM780 MP confocal and multiphoton microscope For a range of experiments from sub-cellular localization to mapping of large tissue sections and in vivo imaging. This system is available for use as part of the MitoCare Shared Resources service center.
405, 458, 488, 514, 561, 594 and 633 nm laser lines for single-photon excitation. Chameleon Vision II system (Coherent) for mulitphoton imaging. The confocal scanner is coupled to a Zeiss inverted microscope (Axio Observer). Three internal detectors including Meta detector for spectral imaging. Two external, non-descanned detectors for multi-photon imaging. Transmitted light detector for DIC/phase contrast. 10x, 20x, 25x, 40x, 63x and 100x objectives. Targeted illumination capability for photo-bleaching/activation; motorized focus for z-stacks; encoded, motorized stage; temperature and humidity controlled incubator with CO2 regulation. Zen acquisition and analysis software.
- High resolution, 3D imaging of live and fixed specimens
- Tile-scan images for large areas
- Imaging in multi-well formats
- Photo-activation, bleaching or other optogenetic techniques
- Deep tissue/in vivo imaging with multi-photon excitation
Zeiss LSM880 with Fast Airyscan has similar confocal capabilities as the LSM780 with updated scanhead and detection. The Fast Airyscan module allows for increased spatial resolution of 1.7x beyond traditional confocal or up to 4x faster recording.
FluoStream
For dynamic measurements of calcium and other biosensors in live cells.
Olympus IX71 inverted microscope coupled with Evolve 512x512 EM-CCD camera, with a back-thinned detector, giving >90% quantum efficiency. Sutter DG4 Xenon light source for rapid excitation selection and motorized filter wheel (ASI FW1000) for emission selection and multiband dichroic mirrors for flexible simultaneous measurements using several fluorophores. Temperature-controlled stage incubator and objective warmer. Operated via custom Spectralyzer software.
MultiFluo
For multi-parameter recording in live cells.
Leica DMI 6000B inverted microscope fitted with motorized turret (400ms switch time among positions) and Sutter DG4 Xenon light source for rapid excitation selection coupled with a EM-CCD camera (ProEM 1024x1024) and temperature-controlled cell perfusion chamber. Operated via custom Spectralyzer software.
FluoTrack
For high-throughput imaging at high resolution in over large areas (tile scan) and/or in multi-well formats.
Olympus IX83 motorized inverted microscope coupled with Sutter DG4 Xenon illumination and Prior high-speed, encoded motorized stage, environment control chamber, hardware autofocus module (ZDC2), and custom multi-band filter sets for long-term, multi-well, multi-color imaging. Back-illuminated sCMOS camera (Hamamatsu Orca Fusion). Operated via MetaMorph software (Molecular Devices).
‘Charlie’
For ultra-high-speed calcium imaging with flash photolysis option.
Olympus IX81 motorized inverted microscope fitted with a Hamamatsu ORCA-Flash 4.0v3 sCMOS camera capable of 100+ frames/s and Hamamatsu W-view Gemini emission splitter for simultaneous imaging of multiple wavelengths or image planes. High-speed excitation switching by Sutter Lambda 421 LED illuminator with 7 wavelength options between 340 and 586 nm and uncaging flash illumination via a high-power 365 nm LED (Sutter TLED+). Prior high-speed, encoded motorized stage, environment control chamber, and hardware autofocus module (ZDC). Operated via Micro-Manager and an Arduino Duo on a PC optimized for rapid data collection and processing.
Ionoptix System
For recording myocyte calcium and contractility events, with ultrafast PMT-based recording at 1000Hz rate.
LIFA-FLIM
(Lambert Instruments)
For quantitatively measuring biosensors independent of their brightness or to measure molecular interactions (by FRET-FLIM) in live cells.
Camera-based system that uses frequency-modulation to rapidly measure fluorophores lifetimes. 445, 488, 561, 640 nm laser illumination and an emission filter wheel allows multi-dye imaging. Operation and lifetime determination via LIFA software.
Disease Modeling
At MitoCare, we also focus on the diagnostics and the pathogenesis of disorders driven by mitochondrial dysfunction.
MitoCare investigators study the consequences of mitochondrial dysfunction at the organismal level (in vivo) and in vitro models.
Every 30 minutes a child is born who will develop a mitochondrial disease by age 10.
Mitocare's Disease Models
- Chronic Alcoholic Rat
- High Fat Diet Mouse
- Friedreich's Ataxia
- PiC Knock-Out
- MICU1 Knock-Out<
- MICU2 Knock Out
- Miro1 Knock Out
- IP3r Knock-Out
- EMRE/Smtd1 Knock-Out
- ACOT2 Knock-Out
- DRP1 Knock-Out
- Fibroblasts and Lymphoblasts
- PiC Loss of Function
- MICU1 and MICU2 Loss of Function
- Autosomal Dominant Optic Atrophy OPA1 mutations
- MSTO1 mutations
- MERRF and MELAS Fibroblasts
- "Common Deletion" Fibroblasts
Using cutting-edge CRISPR Cas9 genetic engineering, MitoCare researchers create new relevant models every day.