Vesi-Ref CD63 GFP
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.
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Vesi-Ref CD63 GFP
Size and concentration measurement
Extracellular vesicles are characteristically broad in size distribution, making the assessment of their size potentially difficult with most analytical methods. Before embarking on a long session, run an aliquot of Vesi-Ref to check the output. Adjust acquisition parameters if size distribution is truncated/not in nominal range or if particle concentration is over/under counted.
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
Vesi-Ref CD63 GFP
Phenotyping by fluorescent labelling
Assessing the sensitivity of detection in fluorescence is crucial, as under or over representation of signal can lead to unsuccessful experiments. Vesi-Ref’s strong GFP signal enables to control the sensitivity of fluorescence detection in most modern techniques aimed at bio-nanoparticles analysis, including:
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)
Application: 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 Technical Description
| Origin | Human embryonic kidney cells (HEK293) |
| Quantity | 100µL (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. |