Journal: Nature protocols

Article Title: High-speed imaging of glutamate release with genetically encoded sensors

doi: 10.1038/s41596-019-0143-9

Figure Lengend Snippet: (a) Left: Setup for affinity and selectivity determination. GEGI in assay buffer is placed into a fluorescence cuvette with a magnetic stirrer and placed inside the sample chamber of the fluorescence spectrometer (Fluorolog3, Horiba Scientific). With an Aladdin pump, the ligand (Glu, Asp, Ser) is continuously added to the cuvette while the fluorescence is recorded (λex = 492 nm, λex = 514 nm). Right: Examples of affinity curves of a GEGI for glutamate (black squares) and aspartate (black circles). The fluorescence emission is corrected for dilution and bleaching and plotted against the glutamate concentration in the chamber. The data is then fitted with a Hill equation (green and orange traces for glutamate and aspartate, respectively). (b) Left: Setup for stopped-flow kinetic measurement. The solutions are rapidly mixed in the mixing chamber and then pushed into the optical cell where the fluorescence is excited at 492 nm and emission is detected by a PMT with a cut-off filter (>530 nm). For association the GEGI in assay buffer without glutamate is loaded into the drive syringe B and mixed with assay buffer containing increasing concentrations of glutamate filled in drive syringe A. For dissociation the GEGI in assay buffer with saturating glutamate concentration is loaded into the drive syringe B and mixed with GluBP 600n in assay buffer filled into drive syringe A. For both measurements the PMT zero level is determined by mixing assay buffer with assay buffer and the intrinsic fluorescence of the GEGI is recorded by mixing the GEGI in assay buffer with assay buffer (both without Glu). Right: Examples for recorded time traces. Top: Fluorescence increase observed when GEGIs are mixed with increasing glutamate concentration. Bottom: Decrease in fluorescence when glutamate is retained from GEGI by GluBP 600n. The raw data are fitted with monoexponential decays (dark green line).

Article Snippet: Reagents Plasmids ○ pCMV iGluSnFR (Addgene plasmid #41732) 9 , iGluSnFR in mammalian expression vector, can be used for expression in HEK293T cells and as starting point for SDM to induce new mutations ○ pET41a(+) (Novagen, Merck cat. no. 70556-3), bacterial expression vector used to express GEGI variants in E. coli ○ pET30b (Merck cat. no. 69909), bacterial expression vector used to express GEGI variants in E. coli ○ pCI syn iGluSnFR (Addgene plasmid #106123), mammalian expression vector to express iGluSnFR in neurons (hippocampal slices) ○ pDisplay FLIPE-600n (Addgene plasmid # 13545) 6 , vector encoding for GluBP 600n for expression of glutamate-binding protein GluBP 600n in E. coli .

Techniques: Fluorescence, Concentration Assay

Journal: Scientific Reports

Article Title: Optical control of filamentation-induced damage to DNA by intense, ultrashort, near-infrared laser pulses

doi: 10.1038/srep27515

Figure Lengend Snippet: ( a ) Gel images obtained after pBR322 plasmid was irradiated with laser light using a 10 cm focal length lens. The negative and positive signs above the image panel indicate, respectively, no laser exposure and laser exposure for 180 seconds. Also shown are schematic depictions of single strand breaks (SSBs) and double strand breaks (DSBs) induced upon laser irradiation. Linear DNA results from DSBs. ( b ) Dependence of the percentage of DNA in supercoiled, relaxed, and linear states on the focal length of the external lens. The bars marked “Control” pertain to DNA prior to irradiation. ( c ) Dependence of the percentage of DNA in supercoiled, relaxed, and linear states for focal lengths of 8.5 cm to 12.5 cm. In each case irradiation was for 180 seconds using 820 nm laser light with the laser energy kept fixed (230 μ J) for different lenses.

Article Snippet: Our DNA (pBR322), obtained from a commercial source (Merck-Millipore, India).

Techniques: Plasmid Preparation, Irradiation, Control