FSEV-AFC Workshop 2024

During the workshop, we will teach you how to use EV kit to immobilize EVs specifically, then do a demonstration how to use SMLM to image EVs to unravel their biomarkers and morphology and finally how to analyze and exploit quantitative data.

Discussion about the best practices for EVs samples preparation and studies by flow cytometry, acquisition and analysis of references sample & material, advantages & benefices of the NovoCyte Quanteon flow cytometer to detect and count small particles.

Flow cytometry can be a powerful and fast technique for Single Extracellular Vesicles (EV) analysis. During this workshop, we will discuss challenges EV researchers might face for robust qualification, quantification and standardization of Single EV analysis as well as how to tackle them.
Starting with a brief description of the BD FACSymphony^TM A1 Cell Analyzer hardware (including the SPD, Small Particle Detector, module) and standardized setup (incl monitoring of low noise levels), we will acquire and review a set of data to better understand the importance of each control/calibrator. Of course, we will also showcase how to relate flow cytometry scatter signals to EV size, using the FlowJoTM Rosetta Calibration plugin along with the Rosetta Calibration beads from Exometry.
Finally, we will also run a “regular” cell sample, showcasing the versatility of the BD FACSymphony^TM A1 Cell Analyzer to run either EVs or cells without the need to change SPD settings.

In the following workshop, we will walk you through use of Violet Side-Scatter and how to use the CytoFLEX to analyze EV and small particles.

During this workshop, we will review best practices to identify, characterize and quantify small particles and extracellular vesicles according to MIFlowCyt-EV standards. We will discuss the sample preparation, instrument calibration and data acquisition and analysis. We will use the Cytek Aurora ESP to show you the acquisition and analysis of reference samples and material.

Based on NTA technology, the ZetaView (Particle Metrix Gmbh) is a fast, robust and versatile device to analyze your EVs.
During the workshop, CD63-eGFP EVs will be used to demonstrate the capabilities of the ZetaView measuring:

• Size and concentration on scatter mode,
• Size and concentration on fluorescence mode,
• Colocalization on fluorescence mode,
• Zeta Potential.

Izon's qEV columns are commonly used for extracellular vesicles (EVs) isolation for many reasons including the rapid and gentle isolation process, the high particle number/µg of protein purity ratio outcome as well as their scalability.
During this workshop, we will show you how your EVs isolation process can be automated with Izon’s Automatic Fraction Collector (AFC) V2 and how you can optimize the process to choose between isolating pure EVs, concentrated EVs, or total EVs recovery. You will not only gain valuable time with the AFC V2, but also reduce operational error and standardise the whole isolation process, making Izon a one-stop shop from EV isolation to characterisation by the advanced Tunable Resistive Pulse Sensing (TRPS) and finally downstream applications including concentration of isolated sample and RNA extraction.

The MACSPlex EV Kit IO (for immuno-oncology) allows the detection and characterization of extracellular vesicles (EVs), using 37 different surface epitopes and two isotype controls. The MACSPlex EV Kit IO comprises a cocktail of various fluorescently labeled bead populations, each coated with a specific antibody binding the respective surface epitopes.
During the workshop we will present the MACSplex kit and how we can analyze it with the MACSQuant analyzer.

Based on the principles of interferometry, the Videodrop makes it possible to measure the concentration & size of your extracellular vesicle solutions:

• in real time (40s),
• in a single drop (5µL),
• without labeling & no purification,
• on viscous & complex samples.

In daily practice, Videodrop can help you optimize several long preparatory steps preventing you from multiple EVs size & concentration characterization compared to traditional methodology. Videodrop appeared an appropriate new method for:

• Fast & reproducible EV quantification,
• High throughput of measures and analysis of a large number of samples on a daily basis,
• Investigations with a limited volume of material,
• Quality control for Bioproduction of EVs used as a therapy.

Illuminate the nanoworld with the NanoAnalyzer!

Underpinned by nano-flow cytometry, the NanoAnalyzer enables quantitative multiparameter analysis of single EVs down to 40nm. Through the measurement of scattered light, statistically robust particle size distribution and concentrations are easily achieved in just 2-3 minutes. Simultaneously, flexible fluorescent labelling strategies can identify EV sample purity, protein presentation, cargo loading and more.

Developing therapeutics using invisible drug delivery systems brings many challenges in biophysical characterization. During process and formulation development, there is a need to monitor sample purity and stability at many points to help guide the next step. Garnering detailed information of these complex systems to fully correlate their composition to their therapeutic effect is also crucial. NanoSight Nanoparticle Tracking Analysis (NTA) has become a standard toolbox in the characterization of biomaterials including extracellular vesicles, drug delivery systems, viral vectors and protein aggregates. NTA provides visual confirmation and high-resolution size and concentration data in minutes allowing the instant assessment of sample stability, but also complexity. With the introduction of the new NanoSight Pro, characterization becomes even easier, quicker and more accurate than before.
Packed with smart features, the NanoSight Pro provides greater sensitivity in biologics detection, high reproducibility and enhanced fluorescence measurements for detecting sample subpopulations. Powered by machine learning algorithms, measurement subjectivity is reduced and automated processing enabled to assure superior Nanoparticle Tracking Analysis.