Assays and Standards
Vesicle Flow Cytometry (VFC)
Vesicle Flow Cytometry (VFC) is a patent-pending approach to quantitating and characterizing individual extracellular vesicles (EVs such as microvesicles or exosomes). Cellarcus’ VFC assays include all standards, calibrators, and other reagents in a kit format to ensure accurate vesicle measurments. VFC leverages strengths of flow cytometry to enable high throughput measurement of vesicle size (down to 70nm), concentration, and surface molecules (down to 30 molecules per vesicle) in quantitative, NIST-traceable units. It is suitable for analysis of purified vesicles, as well as vesicles in diluted biofluids (including plasma, serum, CSF and urine) without further processing.
Traditional flow cytometry which relies on light scatter to detect cells, is not sensitive enough to detect individual vesicles. VFC addresses sensitivity issues using a novel triggering mechanism that greatly enhances the small particle sensitivity of widely available flow cytometers.
You can rely on the accuracy of this assay for critical measurements such as titrating doses of therapeutic vesicle preps. It is the subject of several publications and is validated in 1000s of samples over numerous collaborations with academic and pharmaceutical labs.
What are the recommended flow cytometers for VFC?
VFC requires modern flow cytometers with improved sensitivity over previous generations of lymphocyte analyzers. Currently, the Beckman Coulter CytoFlex™ and Amnis ImageStream™ provide suitably sensitive optics for VFC. We are in the process of qualifying additional flow cytometers for our assay and will include performance information including LOD for vesicle size, concentration, and surface marker immunofluorescence.
How is VFC different from NTA (e.g. NanoSight™)
VFC and NTA are vesicle characterization technologies based on different detection platforms. VFC is a set of reagents including all calibrators and standards designed for analyzing vesicles on modern flow cytometers. NTA is specialized instrumentation for sizing particles that is also used for vesicle analysis.
Because NTA was designed for sizing particles, it has many limitations in vesicle analysis. In general, it is imprecise when estimating vesicle concentration. It also lacks the fluorescence sensitivity to detect antibody labeled vesicles.
VFC solves these problems with concentration estimates that are over 5-fold more precise as well as greatly enhanced fluorescence sensitivity (current limit of detection is 30 molecules/vesicle).
Other advantages of VFC
- Vesicle specificity (NTA measures any particle, not just vesicles)
- Throughput (VFC is capable of processing 100s of samples per day)
How does VFC compare to RPS (resisitive pulse sensing)?
RPS is a much more precise technology for estimating particle size distribution and concentration and can be used for vesicle analysis. It performs equivalently to VFC for vesicle size and concentration estimation, although accuracy of RPS is limited by a lack of vesicle specificity. Compared to VFC, it lacks vesicle specificity and is not suitable for surface cargo measurement.
Synthetic Vesicle Standards
We provide synthetic liposomes of known size distribution as standards for consistent, accurate estimate of vesicle size. Other standards for reporting fluorescence for vesicle surface molecule labeling are in development.
Tissue and Cell Type Specific Vesicle Samples
We offer reference samples from various sample types. These are useful for benchmarking assays, comparing measurements across runs, or for other experiments requiring consistent, well-characterized populations of vesicles from specific sources. All of our vesicle reference samples are characterized via NTA and VFC (including measurement of relevant surface markers) ensuring greatly increased vial-to-vial uniformity vs other companies.
Cargo Specific Positive Control Samples