Computational protocol: An improved monomeric infrared fluorescent protein for neuronal and tumor brain imaging

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Protocol publication

[…] Prior to diffraction experiments, crystals were flash-cooled directly in liquid nitrogen. X-ray diffraction experiments were performed at the European Synchrotron Radiation Facility (Grenoble, France). Data were collected at 100 K at a wavelength of 0.873 Å on beamline ID23-eh2. Diffraction data sets were processed using XDS and intensities were scaled and reduced with AIMLESS . The crystal belongs to the C2 space group and diffracted to 1.14 Å resolution. The solvent content is 46 %. Structure refinement was conducted with Refmac5 using anisotropic B-factors . The thioether bond between Cys24 and the chromophore biliverdin appears to be X-ray sensitive. Structure and experimental data have been deposited in the Protein Data Bank under the PDB ID 4cqh. Crystallographic data statistics can be found in . [...] For characterization in mammalian cells, IFP1.4, IFP2.0, IFP2.0+HO1 and iRFP were cloned into pcDNA3.1 vector under CMV promoter. We also cloned GFP under internal ribosome entry site into the same vector. HEK293A cells were transfected using calcium phosphate transfection method and then imaged 48 h later on a Nikon eclipse Ti inverted epifluorescence microscope with redshifted Cy5.5 filter set (665/45 nm exciter and 725/50 nm emitter, Chroma) and a digital CMOS camera, controlled by NIS-Element software (Nikon Instruments). Images were processed and analyzed with ImageJ. The fluorescence intensity of IFPs was normalized by the fluorescence intensity of co-expressed GFP, to accommodate variations in transfection efficiency among cells.For characterization in neurons, hippocampi were dissected from 19-d embryonic rats (E19), digested with a mixture of proteases at 37°C for 15 min and dissociated with a fire-polished Pasteur pipette in plating medium (minimal essential medium [MEM] containing Earle’s salts with 10% fetal bovine serum, 0.5% glucose, 1 mM sodium pyruvate, 25 μM glutamine, and 1 × penicillin/streptomycin). Neurons were then plated onto glass coverslips (Warner Instruments) pretreated with nitric acid and coated with poly-l-lysine (0.1 mL/mL; Sigma-Aldrich). Each 12-mm coverslip was plated with 5 × 104 neurons, which were maintained in neurobasal medium (Invitrogen) containing B27 extract (Invitrogen), 0.5 mM glutamine, 100 units of penicillin, and 100 μg/mL of streptomycin. Neuronal culture were incubated at 37°C with 5% CO2. For transient transfection, neurons in culture at 9 DIV were treated with Opti-MEM containing IFP1, 4, IFP2.0, IFP2.0+HO1 or iRFP plasmid, and Lipofectamine 2000 (Invitrogen). Images were taken 48h after transfection. Cells were imaged on a Zeiss Axiovert microscope with Cy5 filter set (Chroma) and a CoolSNAP ES2 CCD Camera (Photometrics, Tucson, AZ), controlled by MetaMorph software (version, Molecular Devices, Inc. Sunnyvale, CA). To perform whole-cell recording, recording pipettes were routinely filled with a solution containing (in mM): 125 K- gluconate, 15 KCl, 10 HEPES, 3 MgATP, 0.3 Na-GTP, 5 Na-phosphocreatine and 0.2 EGTA (pH 7.2–7.4, 290–300 mosM). The cells on coverslips were placed in an oxygenated (95% O2 and 5% CO2) solution containing 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose. Action potentials were induced by injection of currents under current clamp. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Mus musculus, Drosophila melanogaster
Chemicals Biliverdine, Oxygen