Recommended reading

Last Update: June 2019

Introduction to Vesicle Flow Cytometry & Standardisation Papers

Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects, Current Protocols in Cytometry, 2015, DOI: 10.1002/0471142956.cy1314s73 This is a great introductory review for researchers wanting to understand the basics of flow cytometry, as well as understand how vesicle flow cytometry differs from cellular flow cytometry, along with the concepts behind fluorescence and scatter standardisation. 

Extracellular Vesicle Flow Cytometry Analysis and Standardization. Frontiers in cell and developmental biology. 2017, DOI: 10.3389/fcell.2017.00078, PMID: 28913335. This is an open access mini-review I wrote, aimed to be good for a short introduction to vesicle flow cytometry and the current problems its utilisation faces in the field. I intentionally included citations to all of the articles I felt were relevant if a researcher wanted to find all the material they need to fully understand the subject. 

Fluorescence Calibration Papers

Wang, L. and Hoffman, R.A. 2017. Standardization, calibration, and control in flow cytometry. Curr. Protoc. Cytom. 79:1.3.1-1.3.27. DOI: 10.1002/cpcy.14. This paper is a good background to understanding what an MESF and ERF unit is and how they can be utilized to calibrate and standardize flow Cytometry data.

Gaigalas AK, Wang L, Schwartz A, Marti GE, Vogt RF Jr. Quantitating Fluorescence Intensity From Fluorophore: Assignment of MESF Values. J Res Natl Inst Stand Technol. 2005;110(2):101–114. Published 2005 Apr 1. doi:10.6028/jres.110.010. This is a technical paper that describes how MESF values are assigned to beads.

Perfetto, S. P., Chattopadhyay, P. K., Wood, J. , Nguyen, R. , Ambrozak, D. , Hill, J. P. and Roederer, M. (2014), Q and B values are critical measurements required for inter‐instrument standardization and development of multicolor flow cytometry staining panels. Cytometry, 85: 1037-1048. doi:10.1002/cyto.a.22579 This is a technical paper that shows the importance of quantifying a flow cytometer’s signal (Q) and background (B) for standardizing data between flow cytometers.

Light Scatter Calibration Papers

Single vs. swarm detection of microparticles and exosomes by flow cytometry, Journal of Thrombosis and Haemostasis. 2012, DOI: 10.1111/j.1538-7836.2012.04683.x, PMID: 22394434. For me, this is a landmark paper in the field for scatter standardisation. This paper was the first to show definitively that polystyrene bead scatter intensity could not be used at face value as a reference material and that their refractive index had to accounted for if being used as a standardisation control. The solution described in this paper the use of scatter modelling. 

Welsh J A, Horak P, Wilkinson J S, Ford V, Jones J C, Smith D C, Holloway J A, Englyst N A, FCMPASS software aids extracellular vesicle light scatter standardisation., Cytometry Part A, DOI: 10.1002/cyto.a.23782. This is a paper I led on using light scatter calibration software (FCMPASS). This paper intentionally gives a basic outline of scattering principles such as why the illumination wavelength, collection angle, and particle composition are all important factors to account for when trying to standardize light scatter. Although this paper covers a technical topic it was purposely written in an accessible manner that minimized complex equations and gave definition of physical phenomena that many biologists may not have heard of. 

EV Standardisation Papers

A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer, Journal of Thrombosis and Haemostasis. 2011, DOI: 10.1111/j.1538-7836.2011.04283.x, PMID: 21481178. This paper was one of the first to show that polystyrene particles over-estimate the diameter of vesicles based on scatter measurements, and that better reference particles were required at a time when flow cytometer scatter modelling for EVs had not been established. This article was contentious at the time due to showing that the community’s attempt at standardising EV flow cytometry, in the form of Megamix beads, was not an accurate sizing method, nor achieved consistent results across different flow cytometry platforms.

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. 2014, DOI: 10.1111/jth.12602, PMID: 24818656. This paper gives a comparison of beads and EVs acquired using TEM, FCM, NTA, and RPS and is useful in comparing not only differences in technique sensitivity but resolution and concentration determination. 

Novel Flow Cytometer Equipment & Methods Papers

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma, Cytometry Part A. 2016, DOI: 10.1002/cyto.a.22621, PMID: 25808430. This is a technical paper that requires a relatively high-level of mathematical and physical science knowledge to fully understand. It however shows the ability to size and derive refractive index by collecting light in a unique manner using a novel flow cytometer and combining it with Mie scattering. 

High sensitivity flow cytometry of membrane vesicles. Cytometry. Part A : the journal of the International Society for Analytical Cytology. 2015, DOI: 10.1002/cyto.a.22787, PMID: 26484737. This paper is one of the earliest to implement fluorescence calibration for vesicle flow cytometry and reports their flow cytometry protocol in a standardised and reproducible manner. In my opinion this article sets the bar for the future of EV standardisation and reporting using flow cytometry. It is also a novel demonstration of inferring EV diameter using fluorescence and an independent EV measuring technique, in this case nanoparticle tracking analysis. 

Light-Scattering Detection below the Level of Single Fluorescent Molecules for High-Resolution Characterization of Functional Nanoparticles, ACS Nano. 2014, DOI: 10.1021/nn505162u, PMID: 25300001. This paper is a demonstration of a next generation high-sensivitiy flow cytometry instrument, that breaks some stereotypes of what is possible using flow cytometry. This instrument claims to be capable of detecting single PE molecules and silica particles <30nm using scatter. It is therefore capable of enumerating the majority, if not all, EVs. This is instrument is now sold as the ‘nanoFCM’.