You can refer to the following links:-

1. http://www.polysciences.com/default/catalog-products/microspheres-particles/flow-cytometry/flow-cytometry-standards/cell-size-estimation/submicron-bead-calibration

2. http://www.polysciences.com/default/catalog-products/microspheres-particles/flow-cytometry/flow-cytometry-standards/cell-size-estimation/

3. http://link.springer.com/protocol/10.1007/978-1-59745-398-1_6

Thanks a lot Sandip Chakraborty for your help. I have attached the BOOK if you ever need it.

Hi Bahaa, 

This very nice article could be helpful for you. Extracellular vesicles cover both exosomes and microparticles (are commonly named as microvesicles, shedding vesicles or ectosomes). 

Below is the link 

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22644/abstract;jsessionid=74C3E3230865BD48C1368B0FC9E228A5.f03t02 

Although this article primarily focuses on sorting, nevertheless you may find some potential information related to your query. 

Best 

Muhammad Nawaz

Many many thanks Muhammad Nawaz

Dear Bahaa,

To be clear, calibration of (absolute) size for EVs/MPs/MVs* is not the task of flows cytometry (FCM). Other technologies [1,2] are more appropriate for doing that e.g. E.M., D.LS., Laser Nanotracking, AFM***, qNano ... Size calibrated (preferably fluorescent) beads are most appropriate to set-up flow cytometers in reproducible conditions in order to reproducibly count "the emerging tip of the EV iceberg" [3,4]. On the other hand, appropriately standardized FCM (using sub-micron beads such as Megamix-Plus and Gigamix, but not only) can help providing numbers that are comparable even if generated on different platforms, as recently illustrated in an international workshop organized under the auspices of ISTH Vasular Biology SSC (Lacroix et al, in preparation). At least, FCM has many advantages over alternative (better sizing) technologies since it provides i) single particle analysis with highly multi-parametric informations linking (immuno-)phenotype with relative size, in addition to being quick, more and more accessible and potentially automatized.

THE BIG ISSUE nowadays is the fact that EVs may not have the same refractive index (R.I.) as polystyrene beads and thus these beads are not optimal size calibration standards. In fact, nobody really knows what the R.I. of EVs is and at least, expert scientists recognize that this will be rather heterogeneous, as are size and composition of EVs. (for review and refs, see 4). This is an actively moving field which you have to regularly follow in the litterature since new data arrive every day/week.

A 2nd BIG ISSUE is to know whether absolute size calibration is really needed in order to validate an EV-based emerging biomarker. Again, this is an open question and most probably informations about phenotype, function, DNA/RNA-based cargo, lipidomics and proteomics will bring more useful data than absolute size calibration alone.  

In any case, I wish welcome to you and any new comer in this highly exciting field ! And to encourage you, please note that from last June (ISAC CYTO 2015 intal meeting in Glasgow, UK), 3 scientific communities decided to join forces to move the field forward more effectively  i.e.  ISAC (Cytometry) + ISEV (EVs) + ISTH (Hemostasis).

*) terminology depends where you are coming from e.g. Hemostasis for MPs, cell biology/Immunology for MVs, ISEV-linked researcher for EVs, the last proposed (possibly official) acronym.

1] Lacroix et al, Overcoming limitations of microparticle measurement by flow cytometry, Semin Thromb Hemost; 36:807-18 (2010)

2] Van der Pol et al, Optical and non-optical methods for detection and characterization of microparticles and exosomes, JTH; 12:2596-607 (2010)

3] Poncelet et al, Standardized counting of circulating platelet microparticles using currently available flow cytometers and scatter-based triggering: Forward or side scatter? Cytometry A, in press doi 10.1002/cyto.a.22685

4] Poncelet et al,Tips and tricks for flow cytometry-based analysis and counting of microparticles. Transfusion and Apheresis Science 53 (2015), pp. 110-126.  doi: 10.1016/j.transci.2015.10.008 --> link for free download until jan 22 (http://authors.elsevier.com/a/1S8cl_XVaVUki9)

Dear Philippe Poncelet

Thanks for the valuable information and the update. In fact, the other technologies for the size calibration are not available in our facility, and the flow cytometer is the only beast choice for my work. Currently, I am the only one who work on Extra Cellular Microvesicles (Platelet extracellular vesicles) in the faculty, and it’s been a kind of challenging of being work alone, especially the field is still considered new. But, I am really positive to achieve my objectives and eager to improve and contribute in this field. And again, Thanks for the welcome and your encouragement its means a lot to me.

Warmest regards

Bahaa

Dear Bahaa, Indeed more and more students, post-docs and even confirmed scientists want to deal with EVs due to their enormous potential as emerging biomarkers. And you are right: FCM is nowadays one the most accessible, quick and powerfuil technique that people tend to try for that purpose, although this has not been initially developped for that specific application.

We have been involved in this field for over 10 years now (at least for Micropaticles/Ectosomes) and realized the dificult task to push our flow cytometers to their real limits. This is the reason why, as a company devoted to create FCM standardized reagents & kits, we also decided to make our optimized tools commercialy available to everybody in the world. These tools are the result of close collaborations with hospital/university research teams also striving to deal REPRODUCIBLY with Microparticles (and nowadays EVs, also see www.isev.org).

My welcome is for you and the many new-comers. Philippe

Note: 3 years ago, Prof. J. Nolan, President of ISAC decided to mount a sub-website with questions related to Microvesicles. http://isac-net.org/Interest-Development-Groups/Microvesicle-Analysis-Interest-Development-Group.aspx  This may also help. As previously stated ISAC and ISEV and also ISTH now tend to synergize to help solving the many questions in this young and expanding field.

Thanks Philippe Poncelet, you have been great help.

Dear Badaa and MP/EV colleagues,

As stated before, BioCytex is striving to provide optimal tools for standardized FCM-based MP analysis and counting. Our last addition to the tool-box are counting beads specially devoted to count MPs: MP-Count beads are 3 µm in diameter, PE-labeled with an intensity which remains compatible with most current MP-adapted protocols and do not present the so-called "fines" i.e. smaller plastic bead debris that contaminate most other available counting beads and enter the scatter-based "MP gate". For an illustration, see my last addition to my RG page (poster at CYTO 2016 on the use of CytoFLEX flow cytometer for MP analysis). Also features Gigamix, a complete mixture of fluorescent beads with 7 different sizes of beads (from 0.1 to 0.9 µm). Again, I need to stress that polystyrene beads are optimal tools for reproducible set-up of scatter settings for standardized MP analysis (Lacroix et al, JTH in press) but not adequate for absolute MP/EV sizing. Hope your work is in good progress! Philippe