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Fluorescence was measured using a SpectraMax microplate reader (Molecular Device) with excitation at 320 nm and emission at 410 nm.
On the basis of our results, we propose that PLD1 is a complex molecular device with functional triad composed of catalytic, PX, and PH domain, each of which senses and mediates discrete steps of EGFR endocytosis (Figure 5E).
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This approach involves molecular manipulation and molecular engineering in the context of building molecular machines and molecular devices with atomic precision.
Glycine buffer (25 μl/well, 0.1 M, pH 10.5) was added to make the contents alkaline, and the absorbance was measured at 570 nm in a multiwell plate reader (Emax, Molecular Devices with Softmax Pro analytical software).
Most importantly, GNR-based nano-devices are expected to behave as molecular devices with electronic properties similar to those of carbon nanotubes (CNTs) [19, 20], as for instance, Biel et al. [21] reported a route to overcome current limitations of graphene-based devices through the fabrication of chemically doped GNR with boron impurities.
Microscopy images were acquired with an ImageXpress Micro microscope (Molecular Devices) with a 10x objective.
Extracellular and cell-associated FITC-Aβ were quantified using an MSF (SpectraMax®, Molecular Devices) with an emission wavelength of 538 nm and an excitation wavelength of 485 nm.
Between each incubation step the wells were washed in PBS using a SkanWasher Plate Washer (Molecular Devices) with four washing cycles of 400 µl each.
Microarrays were immediately scanned at 10 µm resolution in both Cy3 and Cy5 channels with a GenePix 4200AL scanner (Molecular Devices) with a variable PMT voltage to obtain maximal signal intensities with <0.1% probe saturation.
The assays were initiated by addition of pre-activated enzyme and monitored fluorometrically for 30 min with a SpectraMax Gemini fluorescence spectrometer (Molecular Devices) with excitation at 380 nm, emission at 460 nm, and cutoff at 435 nm.
Enzymatic cleavage leading to the release of the fluorescent AMC molecule was measured using a microplate spectrofluorometer (SPECTRAmax Gemini EM, Molecular Devices) with excitation and emission wavelengths of 360 and 440 nm, respectively.
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