405 nm laser

Manufactured by Malvern Panalytical

Sourced in United Kingdom

The 405 nm laser is a compact and efficient light source that emits light in the violet portion of the visible spectrum. It has a wavelength of 405 nanometers and is designed for a variety of laboratory and research applications. The laser's core function is to provide a stable and precise source of monochromatic light.

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405-nm laser instrument LM14 405 nm violet laser unit

15 protocols using 405 nm laser

Nanoparticle Size Determination via NanoSight

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Prior to the NTA analysis, the NanoSight LM10 equipped with a 405nm laser (Malvern Instruments) was calibrated using Silica Microspheres beads (Polyscience). Samples to be measured were then diluted in PBS in order to obtain a particle number between 10⁸-10⁹ particles. At least three repeated-measurements of 60 s were taken per each individual sample and the mean value was used to determine particle number. Static mode (without flow) was used for each analysis. The movement of each particle in the field of view was measured to generate the average displacement of each particle per unit time which was calculated using the NTA 3.0 software.

Borghesan M., Fafián-Labora J., Eleftheriadou O., Carpintero-Fernández P., Paez-Ribes M., Vizcay-Barrena G., Swisa A., Kolodkin-Gal D., Ximénez-Embún P., Lowe R., Martín-Martín B., Peinado H., Muñoz J., Fleck R.A., Dor Y., Ben-Porath I., Vossenkamper A., Muñoz-Espin D, & O’Loghlen A. (2019). Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. Cell Reports, 27(13), 3956-3971.e6.

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Exosome Characterization using NanoSight

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Exosomes (1 × 10⁷ / mL to 1 × 10⁹ / mL) were visualized using a NanoSight NS300 equipped with a 405 nm laser (Malvern, Great Malvern, UK). Exome size and number of particles were assessed. Videos (60 s duration, 30 frames / sec) were recorded and particle movement analyzed using the NTA software (NanoSight version 2.3).

Gao C., Gong Z., Wang D., Huang J., Qian Y., Nie M., Jiang W., Liu X., Luo H., Yuan J., Xiang T., An S., Quan W., Wei H., Zhang J, & Jiang R. (2019). Hematoma-derived exosomes of chronic subdural hematoma promote abnormal angiogenesis and inhibit hematoma absorption through miR-144-5p. Aging (Albany NY), 11(24), 12147-12164.

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Exosome Characterization by TEM, NTA, and ELISA

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Transmission electron microscopy (TEM) was performed to validate exosome
morphology. DPBS-suspended exosomes were deposited on formvar carbon-coated EM
grids. After negative staining with 2% uranyl acetate (pH 4), the grids were
examined and imaged with a FEI TECNAI F20 FEG microscope running at 200 kV of
accelerating voltage, the digital images were recorded with a FEI Eagle CCD
camera (4k x 4k). Images of 100 representative vesicles were measured with
ImageJ (https://imagej.nih.gov). Nanoparticle
Tracking Analysis (NTA) was performed to measure exosomal size and concentration
using a Nanosight LM10 instrument equipped with a 405 nm laser (NanoSight,
Malvern Instruments) at 21°C. The Brownian movement of particles was
tracked by NTA software (version 3.1, NanoSight). Quantitative ELISA assay was
performed to measure exosome marker CD63 [33 (link)] using an ExoELISA-Ultra CD63 kit (System Biosciences Inc., cat.
EXEL-ULTRA-CD63–1) following the manufacturer’s protocol [34 ]. The total protein concentration of
each exosome suspension was measured using NanoDrop_1000 spectrophotometer with
a wavelength 280 nm. DPBS exosome suspensions (normalized to 100 μg of
protein) were plated and run in duplicate. The absorbance of exosomal CD63 was
determined using a Biotek spectrophotometric 96-well microplate reader with a
wavelength of 450 nm.

O’Meara T., Kong Y., Chiarella J., Price R.W., Chaudhury R., Liu X., Spudich S., Robertson K., Emu B, & Lu L. (2019). Exosomal microRNAs associate with neuropsychological performance in individuals with HIV infection on antiretroviral therapy. Journal of acquired immune deficiency syndromes (1999), 82(5), 514-522.

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Quantification and Characterization of sEV

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Protein concentration of sEV samples was assessed employing Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). 9 µL of each sEV sample was lysed with 1 µL of 10× RIPA buffer (Abcam, Cambridge, UK) and incubated for 30 min on a rotating wheel at 4 °C. Samples were then centrifuged at 17,000× g for 20 min at 4 °C. Resulting supernatants were subjected to the BCA assay according to the manufacturer’s instructions. Absorbance was assessed with the use of a MITHRAS LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany). Particle quantification of sEV samples was performed via NTA using NanoSight LM10 equipped with a 405 nm laser (Malvern Instruments, Malvern, UK). For the NTA analysis, samples were diluted 1:500 to 1:1000 in 0.22-µm-filtered PBS. Camera level and detection threshold were set up at 13 and 5, respectively. The absence of background was verified using 0.2-µm-filtered PBS. For each sample, four videos of 60 s each were recorded and analyzed using the NTA 3.0 software version (Malvern Instruments, Malvern, UK).

Bordas M., Genard G., Ohl S., Nessling M., Richter K., Roider T., Dietrich S., Maaß K.K, & Seiffert M. (2020). Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues. International Journal of Molecular Sciences, 21(15), 5586.

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Nanoparticle Size Analysis of In Vitro Vesicles

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Sizes of vesicles budded in vitro were estimated using the NanoSight NS300 instrument equipped with a 405-nm laser (Malvern Instruments, Malvern, UK). Particles were analyzed in scatter mode without a filter. 100-nm silica microspheres (Polysciences, Warrington, PA, USA) were analyzed to check instrument performance and determine the viscosity coefficient of B88. Aliquots (1 μl) of vesicles diluted 1000× with 999 μl of filtered B88 (0.02 μm, Whatman). The samples were automatically introduced into the sample chamber at a constant flow rate of 50 (arbitrary manufacturer unit, ∼10 μl/min) during five repeats of 60-s captures at camera level 11 in scatter mode with Nanosight NTA 3.1 software (Malvern Instruments). The particle size was estimated with detection threshold 5 using Nanosight NTA 3.1 software, and then “experiment summary” and “particle data” were exported. Particle numbers in each size category were calculated from the particle data, in which “true” particles with a track length of more than 3 were pooled, binned, and counted with Excel (Microsoft).

Melville D.B., Studer S, & Schekman R. (2020). Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly. The Journal of Biological Chemistry, 295(25), 8401-8412.

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Nanoparticle Tracking Analysis of Extracellular Vesicles

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The number of vesicles recovered was determined by Nanoparticle Tracking Analysis (NTA) on a Nanosight LM-10HS equipped with a 405nm laser (Malvern) that was calibrated with polystyrene latex microbeads at 100 nm and 200 nm prior to analysis. Resuspended vesicles were diluted with PBS to achieve between 20–100 objects per frame. EVs were manually injected into the sample chamber at ambient temperature. Each sample was measured in triplicate at camera setting 13 with acquisition time of 30 s and detection threshold setting of 7. At least 200 completed tracks were analyzed per video. The NTA analytical software version 2.3 was used for capturing and analyzing the data.
The morphology of the EV isolated were assessed using transmission electron microscopy (TEM) as previously described [15 ].

Akers J.C., Ramakrishnan V., Yang I., Hua W., Mao Y., Carter B.S, & Chen C.C. (2016). Optimizing Preservation of Extracellular Vesicular miRNAs Derived from Clinical Cerebrospinal Fluid. Cancer biomarkers : section A of Disease markers, 17(2), 125-132.

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Rapid Exosome Characterization by NTA

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For the rapid in vitro measurements of exosomes, we performed Nanoparticle tracking analysis (NTA) using NanoSight NS500 equipped with an sCMOS Trigger camera and a 405 nm laser (Malvern Instruments Ltd., Malvern, UK). NTA utilizes the properties of both light scattering and Brownian motion to obtain the size distribution and concentration measurements of particles in liquid suspension. The measured data were analyzed using NTA2.3 analytical software. Each sample was diluted in PBS before the measurements to optimize the number of particles. Samples were measured in quintuplicate in 60-s videos with manual shutter and gain adjustments.

Takacova M., Barathova M., Zatovicova M., Golias T., Kajanova I., Jelenska L., Sedlakova O., Svastova E., Kopacek J, & Pastorekova S. (2019). Carbonic Anhydrase IX—Mouse versus Human. International Journal of Molecular Sciences, 21(1), 246.

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Nanoparticle Size Estimation Protocol

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Sizes of vesicles budded in vitro were estimated using a NanoSight NS300 instrument equipped with a 405-nm laser (Malvern Instruments, Malvern, United Kingdom). Particles were analyzed in the scatter mode without a filter. Silica 100-nm microspheres (Polysciences, Warrington, PA) were analyzed to check instrument performance and determine the viscosity coefficient of B88. Aliquots (20 μl) of vesicles were collected from the top of the flotation gradient as described in the vesicle budding reaction section and diluted 50× with 980 µl filtered B88 (0.02 µm; Whatman). The samples were automatically introduced into the sample chamber at a constant flow rate of 50 (arbitrary manufacturer unit, ∼10 µl/min) during five repeats of 60-s captures at camera level 11 in scatter mode with Nanosight NTA 3.1 software (Malvern Instruments). The particle size was estimated with detection threshold 5 using the Nanosight NTA 3.1 software, after which “experiment summary” and “particle data” were exported. Particle numbers in each size category were calculated from the particle data, in which “true” particles with track length >3 were pooled, binned, and counted with Excel (Microsoft).

Melville D., Gorur A, & Schekman R. (2019). Fatty-acid binding protein 5 modulates the SAR1 GTPase cycle and enhances budding of large COPII cargoes. Molecular Biology of the Cell, 30(3), 387-399.

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Nanoparticle Tracking Analysis of EV Size

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EV size distribution profiles and concentration measurements were obtained by nanoparticle tracking analysis (NTA), using a NanoSight LM14c instrument equipped with a 405 nm laser (Malvern) and NTA software version 3.1 (Malvern). Silica 100 nm microspheres (Polysciences, Inc.) were routinely analyzed to check instrument performance (Gardiner et al., 2013 (link)). NTA acquisition and post-acquisition settings were optimized and kept constant for all samples. These settings were established using Silica 100 nm microspheres (Gardiner et al., 2013 (link)) and subsequently adjusted for optimal detection of MSC-EVs.
EV samples were diluted in 2 mL of PBS 1x in UltraPure^TM DNase/RNase-Free Distilled Water, to obtain a final concentration in the range of 5 × 10⁸ to 3 × 10⁹ particles/mL. Samples were measured using a camera level of 13. Acquisition temperature was controlled and maintained at 20°C. Each sample was recorded 10 times for 30 s, using fresh sample for each acquisition (by pushing the sample syringe). The detection chamber was thoroughly washed with PBS between each sample measurement. A threshold level of 7 was applied for video processing. Each video recording was analyzed to obtain the size and concentration of EVs.

de Almeida Fuzeta M., Bernardes N., Oliveira F.D., Costa A.C., Fernandes-Platzgummer A., Farinha J.P., Rodrigues C.A., Jung S., Tseng R.J., Milligan W., Lee B., Castanho M.A., Gaspar D., Cabral J.M, & da Silva C.L. (2020). Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System. Frontiers in Cell and Developmental Biology, 8, 553444.

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Nanoparticle Size and Concentration Analysis

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The NanoSight NS500 instrument equipped with a 405 nm laser (Malvern Panalytical Ltd, Malvern, UK) was calibrated using Silica Microspheres beads before measurements. Samples were diluted 1/1000 in PBS in order to obtain a particle concentration between 10⁸ and 10⁹ particles/mL. Three repeated measurements of 60 s were taken per sample and the mean value was used to determine particle number. The temperature of the laser unit was controlled at 25 °C. NTA software measured the size distribution (ranging from 10 to 1000 nm) and concentration (particles/mL) of nanoparticles.

Rodríguez-Cobos J., Viñal D., Poves C., Fernández-Aceñero M.J., Peinado H., Pastor-Morate D., Prieto M.I., Barderas R., Rodríguez-Salas N, & Domínguez G. (2021). ΔNp73, TAp73 and Δ133p53 Extracellular Vesicle Cargo as Early Diagnosis Markers in Colorectal Cancer. Cancers, 13(9), 2240.

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