EV core

Nanoparticle tracking analysis (NTA) is a method used to quantify and determine the size distribution of nanoparticles ranging from 70 nm to 2 µm of diameter, depending on the instrument. The sample is injected to the measuring chamber and the sample particles are visualized using a laser beam. Particles passing through the beam are seen as small points of light moving rapidly under Brownian motion, allowing information on particle properties to be obtained. EV-Core currently has two NTA instruments, which compliment each other in terms of accuracy of particle concentration measurements, size measurements and zeta potential measurements. 

The Nanopartice Tracking Analysis provides characterization of:

• Extracellular vesicles
• Microparticles
• Nanoparticles

Particle Metrix Zetaview PMX-120

The ZetaView (PMX-120) is a new-generation NTA technology, which can measure particle size, particle concentration and zeta-potential with a single injection of the sample. The instrument can be operated in scatter and fluorescent modes to measure unlabelled and fluorescently labelled samples. The EV Core currently provides measurement services and training for scatter mode only. 

Particle Metrix Zetaview instrument in a nutshell 

• Measurements are done at 11 positions, which enables a more robust measurement 
• Automated auto-alignment and auto- focus functions to optimize the optical set-up
• Required sample volume is 1 ml, pre-diluted in 0.1 μM filtered Milli-Q water  
• Detection range is 70-2000 nm depending on the type of sample and the measuring mode 
• Optimal concentration range is approximately 105 – 109 particles/ml 
• Continuous and pulsed modes available for Zeta-potential measurements depending on the conductivity of the sample 
• Fast built-in data processing, with several post-acquisition parameters that can be adjusted without having to repeat the measurement 
• Results are provided in PDF and excel files

Service for Zetaview PMX-120

Minimum charge is for 1 h, after which every starting ½ hour is charged. Preparing the instrument (e.g. auto-alignment) prior to the first measurement takes 10 to 15 minutes. Auto-alignment is carried out with 100 nm polystyrene beads before the measurements. Milli-Q water and reference beads are provided by the EV-Core. Measuring one sample takes approximately 1-3 minutes (when dilution factor and optimal parameters are decided). Flushing the measurement chamber with 10-20 mL of Milli-Q water for 1-2 minutes is required between samples, after which the sample is non-recoverable. 

Note, that the ZetaView NTA can only be booked by trained users and is set up for scatter mode only. Service will be available for fluorescent samples later in 2021. 

Spectradyne nCSI: Microfluidic resistive pulse sensing (MRPS)

Spectradyne nCS1 uses microfluidic resistive pulse sensing (MRPS), which uses electrical sensing to measure the diameter of each particle as it passes through a nanoconstriction. It thus provides sizing and concentration information on nanoparticles.  

Spectradyne nCS1 instrument in a nutshell

• Sample requirement is 3 ul for single-use microfluidic cartridges
• Particle size analysis ranges from 50 nm to 10 μm in diameter
• Optimal concentration range is 1 x 104 – 5 x 1011 particles/mL (depending on the cartridge size)
• Preparations take less than half a day and measurement time is 2-6 minutes for each sample.  

nCS1TM service will be available in the EV-Core in autumn 2021. Read more from the manufacturer’s website. 

qNano -Tunable Resistive Pulse Sensing (TRPS) 

qNano uses tunable resistive pulse sensing (TRPS) where particles passing through a nanosized pore cause temporary decreases in electronic current. Detection of the magnitude and frequency of these blockages enable nanoparticle counting and sizing. Access to qNano instrument is through collaboration with Biocomplex core facility at the University of Helsinki. 

qNano instrument in a nutshell

• ‍Particle size analysis ranges from 40 nm to 11,3 μm in diameter and is based on selecting the correct nanopore
• 12 different nanopore options, which need to be purchased separately
• qNano requires 35 ul of sample
• Optimal concentration is 2 x 109  - 1 x 1010 for EV measurements depending on the size of the used nanopore.
• Preparations take approximately one hour and optimally, the measurement time for one sample and calibration beads is approximately 15 minutes. 

qNano measurements will be available at the EV-Core in autumn 2021. Read more from the manufacturer’s website. 

ExoView R100 provides a single particle interferometric reflectance imaging sensor platform (SP-IRIS) for detection of markers on EVs and particle sizing. Complex biological medias such as cell culture and blood serum can be measured according to the manufacturer, but in some cases SEC isolation of EVs is advised. ExoView is a chip-based platform with multi-level measurements, that provide information on:

• Particle size
• Relative particle count
• Biomarker distribution as detected by the chosen antibodies 
• Biomarker colocalization on the particle surface

The imaging platform uses single-use microarray chips. EVs expressing specific surface markers bind to specific antibody spots on microarray chips from where they can be characterized by the choice of antibodies in the detection fluid with up to 3 fluorescent antibodies. Different kits are available for different sample types, and need to be purchased separately.

ExoView R100 instrument in a nutshell: 

• Single-use chip-based platform with multiple antibody customization options
• Colocalization of up to 3 different antibodies
• Tetraspanin kit for cell culture contains CD9, CD63 and CD81 as capture antibodies and mouse IgG as isotype control
• Plasma kit contains CD9, CD63, CD81 and CD41a as capture antibodies and IgG as isotype control
• Three technical replicates and a quality check for each sample are performed in one measurement
• Size detection from 50 nm upwards
• 35 ul of diluted sample (at least 1:1) and optimization of the sample dilution is required
• Assay takes 2 days, with a measurement time of ~10 minutes for each chip 
• Data is provided as a pdf report and can be exported as excel or image files

ExoView R100 service will be available at the EV-core in autumn 2021.

The Apogee A50-Micro flow cytometry is specifically designed for measuring sub-micron biological particles. In contrast to conventional flow cytometers that have difficulties in detecting sub-micron particles, Apogee A50-Micro can measure biological particles down to approximately 200 nm in diameter, thus providing research opportunities in the study of e.g:

• Extracellular vesicles
• Nanoparticles
• Platelets

Apogee’s light scatter and fluorescence detectors are routinely calibrated, which enables improved data-analysis and comparison of data between laboratories and instruments. With the calibrated Apogee A50-Micro one can:

• Measure EV concentrations
• Size EVs
• Characterize EVs via fluorescent labeling (single- and double-marker analyses)
• Quantitate fluorescence

Apogee A50 instrument in a nutshell

• Three lasers: violet (405 nm), blue (488 nm) and red (638 nm)
• Six fluorescent detectors: 405-green, 405-orange, 488-green, 488-orange, 488-red, and 638-red
• Two light scatter detectors: 405-FSC and 405-SSC
• Two sample formats: manual (1.5 ml tubes) and autosampler (96-well plate)
• Minimum sample volume: 200 µl
• Optimal particle concentration: 107-108/ml
• File format: .fcs (data can be exported to excel format)

Service

Minimum charge is 1 h, after which every starting ½ hour is charged. The minimum time includes two sets of control beads and the baseline buffer run. Running one sample takes typically 2-5 minutes followed by 1 minute flushing cycle. The washing steps after samples are stringent and take from 20 minutes to 1 hour, depending on the number and concentration of samples. 

Basic solutions and disposables are provided. In case the user is not providing fluorescently labelled probes please contact the EV core for possibility to use EV-unit’s probes for additional fee.

Electron microscopy (EM) is one of the basic characterization techniques used to verify the success of EV isolation and to detect specific antigens such as EV marker proteins directly in situ. EM allows visualization and quantification of the morphology, size distribution and labelling of the EVs. It also gives information of the purity of EV preparations.

Service

FIMM HiPrep Extracellular Vesicles Core provides EM and immuno-EM sample preparation using whole mount samples and negative staining protocol and, optionally, microscopy of the EV samples.

Basic solutions and disposables are provided. The suspension buffer of the EV sample should preferably be filtered with 0.1-0.2 µm filter to deplete any external particles in the sample.

EV core facility collaborates with the EM unit, Institute of Biotechnology, Viikki.

Email: ev-core@helsinki.fi
Location: Viikinkaari 9, Biocenter 1, 00790 Helsinki, Finland 

Personnel

• Head of the EV core Viikki, Pia Siljander, pia.siljander|at|helsinki.fi
• Technical assistant, EV core Viikki, Helin Veskiväli, helin.veskivali|at|helsinki.fi 
• Technical assistant, EV core Viikki, Rosabella Hartman, rosabella.hartman|at|helsinki.fi
• Head of the HiPrep core (FIMM), Maija Puhka, maija.puhka|at|helsinki.fi