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Biology

FRET Imaging no hidrogéis tridimensionais

Published: August 1, 2016 doi: 10.3791/54135
Automatic Translation
*1, *1,3, 1,2
1Department of Oral Biology, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 2Department of Bioengineering, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 3Laerdal AS
* These authors contributed equally

Materials

Name Company Catalog Number Comments
Polydimethylsiloxane (PDMS) [Sylgard 184 Elastomer Kit] Fisher Scientific (Ellsworth Adhesives) NC0162601 Reagents
Polyethylene glycol dimethacrylate (PEGDM) Prepared according to Lin-Gibson with PEG from Sigma Aldrich 95904 Reagents
3-(Trimethoxysilyl)propyl methacrylate Sigma Aldrich M6514 Reagents
3-glycidoxypropyltrimethoxysilane Sigma Aldrich 440167 Reagents
Dimethyl Phenylphosphonite Sigma Aldrich 149470-5g Reagents
2,4,6-trimethylbenzoyl Chloride Fisher Scientific AC244280100 Reagents
2-Butanone Fisher Scientific AC149670010 Reagents
Lithium Bromide Fisher Scientific AC199871000 Reagents
Isopropanol Sigma Aldrich 190764 Reagents
Sigmacote (siliconizing reagent) Sigma Aldrich SL2 Reagents
Toluene  Fisher Scientific AC36441-0010 Reagents
Phosphate Buffered Saline (PBS) Hyclone SH3025601 Reagents
Opti-MEM Reduced Serum Medium, no phenol red (phenol- and serum-free medium) Life Technologies 11058-021 Reagents
FuGENE HD Transfection Reagent (transfection reagent) Promega E2311 Reagents
Cellstripper (cell dissociation solution) Corning 25-056-CI Reagents
Growth Factor Reduced Matrigel, Phenol Free Corning 356231 Reagents
Forskolin Sigma Aldrich F6886 Reagents
Pipette Tips (10,200,1,000 μl)  Fisher Scientific 02-707-474, 02-707-478, 02-707-480 Disposable lab equipment
Powder Free Examination Gloves Fisher Scientific 19-149-863B Disposable lab equipment
Aluminum foil Fisher Scientific 01-213-100 Disposable lab equipment
Biopsy punch (3 mm) Miltex 3332 Disposable lab equipment
Glass coverslips Fisher Scientific 12-544C Disposable lab equipment
Precoated glass coverslips (epoxysilane) Arrayit CCSL Disposable lab equipment
Glass slide Fisher Scientific 12550D Disposable lab equipment
Sterile vials (15 ml, 50 ml conical) Fisher Scientific 14-959-70C, 14-959-49A Disposable lab equipment
Cell culture dish (6-well) Falcon 351146 Disposable lab equipment
Epifluorescent Microscope with motorized stage Nikon MEA53100 (Eclipse TiE Inverted) Large/non-disposable lab equipment
Adjustable Specimen Holder for XY Stage Nikon 77011393 Non-disposable lab equipment
60X (1.3 NA) objective (CFI Plan Apochromat 60XH Lamda) Nikon MRD01605 Non-disposable lab equipment
CFP/YFP light source and excitation / emission filter holders (Lambda light source & wheel, Lambda emission filter system) Nikon (Sutter Instrument reseller) 77016231, 77016088 Non-disposable lab equipment
CYF/YFP filter set Nikon (Chroma Technology Corp) 77014928 Non-disposable lab equipment
Camera (ORCA Flash 4.0 v1) Nikon (Hamamatsu reseller) 77054076 Non-disposable lab-equipment
NIS-Elements 40.20.02 microscope software) Nikon MQS31100  Non-disposable lab equipment
Prism (spreadsheet and graphing software) GraphPad Software, Inc Version 6 Non-disposable lab equipment
OmniCure S1000 UV Spot Curing Lamp System with 365nm filter or other light source (λ = 365 or 405) OmniCure S1000 Large/non-disposable lab equipment
Pipette Aid  Drummond Scientific Co.  DP-100 Non-disposable lab equipment
Hemacytometer Hausser Scientific Reichert Bright-Line 1475 Non-disposable lab equipment
Vacuum desiccator chamber/degasser  Any Large/non-disposable lab equipment
Tissue Culture Hood  Any Large/non-disposable lab equipment
Chemical Fume Hood  Any Large/non-disposable lab equipment
Oven Any Large/non-disposable lab equipment
Spatula Any Non-disposable lab equipment
Beaker Any Non-disposable lab equipment
Incubator  Any Large/non-disposable lab equipment

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References

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FRET Imaging no hidrogéis tridimensionais
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Cite this Article

Donius, A. E., Bougoin, S. V.,… More

Donius, A. E., Bougoin, S. V., Taboas, J. M. FRET Imaging in Three-dimensional Hydrogels. J. Vis. Exp. (114), e54135, doi:10.3791/54135 (2016).

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