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Vesi-Ref CD63-GFP: Fluorescent extracellular vesicle standard for nano-bio research.

Calibration of nanoparticle measurement.

Vesi-Ref CD63-GFP introduces certainty and repeatability in functional nanoparticles analysis. By assessing the limits of detection of analytical instruments used to measure size, concentration and biomarkers by fluorescence labelling, it is possible to confirm that unknown samples are correctly characterised for these properties.

Nano-Flow Cytrometry Analysis

Size & Concentration Measurement

  • NTA – Nanoparticle Analysis
  • DLS – Dynamic Light Scattering
  • nFCM – Nano-Flow Cytometry
  • TEM – Transmission Electron Microscopy
  • SP-IRIS – Single Particle Interferometric Reflective Image Sensing
  • TRPS & MRPS – Resistive Pulse Sensing
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Phenotyping by Fluorescent Labelling

  • Flow Cytometry (cell analyzers and FACS sorters)
  • Fluorescence Nanoparticle Tracking Analysis (f-NTA)
  • Single Particle Interferometric Reflectance Image Sensing (SP-IRIS)
  • Nano-Flow Cytometry (nFCM)
  • Fluorescence-Microfluidic Resistive Pulse Sensing (F-MRPS)
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Vesi-Ref CD63 GFP Cell Tracking: Fluorescent extracellular vesicle standard for nano-bio research

Application: Data from Hyperion

Application: Data from Unchained Labs

Applications

Positive control of tetraspanin expression in F-MRPS measurement with Vesi-Ref CD63-GFP

Fluorescence Microfluidic Resistive Pulse Sensing (F-MRPS, Spectradyne) was used to measure Vesi-Ref CD63-GFP without modification and after staining with PE-anti-CD63.

F-MRPS detects and measures the size and concentration of nanoparticles using MRPS, an electrical technique, while simultaneously measuring the fluorescence of each particle in up to 3 optical bands. Particle detection and fluorescent measurements are triggered using the MRPS signal, ensuring that each event represents a physical particle larger than the size limit of detection of the cartridge used (65 nm in this case). A one-time factory cross-calibration of the instrument to NIST-traceable MESF standard beads enables particle brightness to be reported by default in traceable units (ERF or MESF when appropriate).

The main figure shows particle concentration vs. size for all particles in the unstained sample (purple), all particles in the stained sample (blue), and subpopulations of the stained sample after gating GFP+ (green), CD63+ (yellow), and double-positive GFP+/CD63+ (red) populations. While the overall particle size distribution displays an approximate power-law dependence of concentration on size that extends to the lower size limit of detection for this cartridge, the fluorescent subpopulations each show the presence of a peak near 75 nm diameter.

The 2D scatter plot (inset) shows the brightness of each particle in FITC and PE channels. Absolute concentration and relative abundance of particles are reported for each quadrant, the bounds of which are defined by the fluorescence limits of detection of each channel: 30 FITC ERF and 2.5 PE MESF.

Vesi-Ref CD63 GFP: Fluorescent extracellular vesicle standard for nano-bio research
Vesi-Ref CD63 GFP: Fluorescent extracellular vesicle standard for nano-bio research

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Application: positive control of tetraspanin expression in F-MRPS measurement with Vesi-Ref CD63-GFP

Vesi-Ref CD63-GFP Technical Description

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OriginHuman embryonic kidney cells (HEK293)
Quantity100µL (1×109 particles)
Storage conditions (lyophilised)Up to 1 years at 4°C
Storage (reconstituted suspension)Up to 6 months at -80°C. Up to one month at -20°C.

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