Advanced Technologies for Research

 

With NEI, other non-federal and Departmental support, we have established a laboratory that has the equipment and personnel to successfully perform the proposed research. The Corneal Neurobiology Laboratory is located in the UIC College of Medicine Research Building (COMRB) - a state-of-the-art facility with an open floor plan to promote collaboration and interaction among scientists. Laboratories of our collaborators in neuroscience (Dr. Lazarov) and hematology (Dr. Rondelli) are also located in the COMRB. Three NEI funded cornea service researchers occupy laboratory space adjacent to Corneal Neurobiology Laboratory; this facilitates collaboration and free exchange of ideas.

The Corneal Neurobiology Laboratory occupies 600 sq. ft. of main laboratory area and has a dark room for imaging that measures 200 st. ft. Additionally there is a 125 sq ft office for the PR. The laboratory has 6 desks with computers on each (Intel Core i7 CPU 870 @ 2.93GHz, with 12 GB RAM, 64 bit operation system). To enable collaborative work, each computer has access to a shared network drive as well as a shared Dropbox folder.

Corneal Neurobiology Laboratory Equipment

Imaging platforms of Corneal Neurobiology Laboratory are housed in a adjacent dark room. The PI has unrestricted access to these equipments. These state-of-the-art platforms include:

1. For 2D in vivo imaging: SteREO Lumar.V12 (Carl Zeiss) with Axiovision software, MRm camera with 8x maximum magnification. We have used this imaging platform to perform wide-field imaging of thy1-YFP mouse corneas to determine nerve fiber density on maximum intensity projection images.

2. For 3D in vivo imaging: Axio Zoom.V16 (Carl Zeiss) with ApoTome.2, Zen software and MRm camera with 25x maximum magnification. ApoTome.2 will enable structured illumination optical sectioning. 3D reconstruction will be performed using the Zen module. In Specific Aim 1, we will perform correlative microscopy to study YFP+ cell interaction with regenerating corneal nerves.

3. For in vitro cell imaging: Axio Observer.Z1m (Carl Zeiss) with incubator for time lapse studies. Lens objectives include: long distance 5x, 10x, 20x, 40x for imaging YFP+ neurites of trigeminal ganglion cells in compartmental cultures in plastic dishes. 40x and 63x water-immersion objective are also available. Filters available are: Cy3, YFP, GFP, DAPI, DIC and BF.

4. For image analysis: Imaging workstation with Neurolucida and AutoNeuron software (MicroBrightField Bioscience, Williston, VT)

Major equipment for molecular analysis that are housed in the dark room of Corneal Neurobiology Laboratory include:

- ODYSSEY Infrared Imaging System (LI-COR Biosciences, U.S.) for quantitative western blot analysis.

- Molecular Imager Gel Doc™ XR+ System with Image Lab™ Software (Bio-Rad Laboratories Ltd.) to image segregated DNA strands after Gel Electrophoresis.

- Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.) for quantitative assays - StepOnePlus™ Real-Time PCR System (Applied Biosystems).


Other general equipment in the Corneal Neurobiology Laboratory include: (i) Refrigerators/Freezers to maintain temperature requirements (40c/ -200c/ -800c), (ii) refrigerated Eppendorf Centrifuges (5810R, 5417R) with maximum rpm of 4000 and 14000 rpm, respectively, and a capacity of 4*1.1 kg, 30*3.75gms respectively. (iii) Hot Rock N Roller Heated Platform Rocker (Thomas Scientific, Swedesboro, NJ, USA). (iv) ProBlot™ 25XLD Rocker with double platform (Labnet International, Inc.). (v) Precision 180-Series Water Bath (Thomas Scientific). (vi) Vortex Genie 2 (Scientific Industries). (vii) Magnetic stirrers (Labworld). (viii) Mini plate spinner (Labnet). (ix) Surgical operating microscope.

Shared equipment in COMRB cornea laboratory space include: Two cell culture rooms with 2 laminar flow hoods (BioGard Hood, the BAKER Company, Inc. and Nuaire, Biological Safety Cabinets), 2 incubators and Water Bath (Precision Stainless Steel Model 183) and a 4 degrees cold room.

UIC Department of Ophthalmology Core equipment:

Department of Ophthalmology Core equipment that will be used in our R01 grant include: (i) Excimer laser (Nidek) for performing annular keratectomy in mouse corneas. (ii) Confocal imaging system: inverse Axio Observer , LSM 710 (Carl Zeiss). (iii) Cell observer SD, Spinning Disk technology from Yokogawa CSU-X1 (Carl Zeiss). Live cell imaging with confocal microscopy using dual camera technology with Zen Software.

UIC College of Medicine Core equipment:

For research proposed in our R01 application we will use equipment housed in the following cores:

(i) Flow Cytometry Service (FCS). The FCS maintains a traditional cell sorter the Beckman-Coulter Elite ESP, a high speed sorter the Beckman Coulter Legacy MoFlo and a Bio-Rad Bio-Plex flow cytometer. Each instrument is capable of analyzing and sorting (Elite and MoFlo) thousands of cells/particles per second. Five to eleven parameters (relative size, granularity, and up to three/nine colors of emitted fluorescence) can be measured simultaneously and correlated particle by particle. We are collaborating with Balaji Ganesh PhD, Director of FCS for performing flow cytometry experiments proposed in this grant.

(ii) Molecular Interactions Laboratory (MIL). The MIL houses Biacore T100, which is the most up to date surface plasmon resonance instrument (SPR) available for real time monitoring of protein-protein interactions and protein-small molecule interactions. We will use SPR analysis in Specific Aim 3. The T100 enables the measurement of binding affinity and binding kinetics, concentration determinations, binding specificity analyses and epitope mapping.

(iii) Protein Research Laboratory (PRL). In Specific Aim 3, we require several peptides spanning RGD and/or RTS integrin binding motifs of sema7a. PRL services will be used for peptide synthesis and purification. We have an ongoing collaboration with Bao-Shiang Lee PhD, Director of PRL. Dr. Lee will synthesize the peptides.