Learning Resources | Flow Cytometry | Useful Articles

Below is a concise list of publications that may be useful when entering the small particle flow cytometry field.

2020

First demonstration of quantitatively calculating light scatter resolution using light scatter modelling.
de Rond L, Coumans F A W, Welsh J A, Nieuwland R, Leeuwen T G., van der Pol E, Quantification of light scattering detection efficiency and background in flow cytometry., Cytometry Part A, doi: 10.1002/cyto.a.24243 [Website]

Review outlining considerations when measuring and reporting EV characteristics of size, concentration, refractive index, and epitope abundance.
Welsh J A, van der Pol E, Bettin B A, Carter D R F, Hendrix A, Lenassi M, Langlois M, Llorente A, van de Nes A S, Nieuwland R, Tang V, Wang L, Witwer K W, Jones J C, Towards defining reference materials for extracellular vesicle refractive index, epitope abundance, size, and concentration., Journal of Extracellular Vesicles, doi: 10.1080/20013078.2020.1816641 [Website]

Position paper providing single EV flow cytometry reporting framework and minimum experimental guidelines.
Welsh J A, van der Pol E, Arkesteijn G, Bremer M, Brisson A, Coumans F, Dignat-George F, Duggan E, Ghiran I, Giebel B, Görgens A, Hendrix A, Lacroix R, Lannigan J, Libregts S, Lozano-Andrés E, Morales-Kastresana A, Robert S, de Rond L, Tertel T, Tigges J, de Wever O, Yan X, Nieuwland R, Wauben M, Nolan J, Jones, J C., MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments, Journal of Extracellular Vesicles. doi: 10.1080/20013078.2020.1713526 [website]

First cross-platform comparison of fluorescence, light scatter, and concentration calibration showing its utility across instrument settings and different flow cytometers.
Welsh J A, Jones J.C. Tang V A., Light scatter and fluorescence calibration allow standard comparisons of small particle data between different instruments, BioRxivs, doi: 10.1101/796961. [website] [cytometry files]

2019

Outlines the characteristics of polystyrene, silica, and EV particle light scattering. First release of FCMPASS software.
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 [website]

2018

First EV flow cytometry standardization study to use light scatter calibration.
van der Pol E., Sturk A., van Leeuwen T., Nieuwland R., Coumans F., ISTH‐SSC‐VB Working group Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. J Thromb Haemost 2018; 16: 1236– 1245, DOI: 10.1111/jth.14009 [website]

Paper outlining the options and consideration when performing light scatter calibration.
de Rond, L., Coumans, F. A., Nieuwland, R., van Leeuwen, T. G., & van der Pol, E. Deriving extracellular vesicle size from scatter intensities measured by flow cytometry Current Protocols in Cytometry, 86, e43. doi: 10.1002/cpcy.43 [website]

Paper demonstrating the use of refractive index and sizing of EVs using the ratio of forward and side light scatter measurements.
van der Pol E., de Rond L., Coumans F. A.W., Gool E L., Böing A. N., Sturk A, Nieuwland R, van Leeuwen T.G., Absolute sizing and label-free identification of extracellular vesicles by flow cytometry Nanomedicine, doi: 10.1016/j.nano.2017.12.012 [website]

This is the second installment of the MISEV guidelines and is useful for knowing the nomenclature and orthogonal assays expected of EV studies.
Théry C., Witwer K., et al., Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines , Journal of Extracellular Vesicles, 8:1, 1535750, doi: 10.1080/20013078.2018.1535750. [website]

2017

Short review on EV flow cytometry pitched at a beginner level.
Welsh J.A., Holloway J.A., Wilkinson J.S., Englyst E.A., Extracellular Vesicle Flow Cytometry Analysis and Standardization. Frontiers in cell and developmental biology. 2017, DOI: 10.3389/fcell.2017.00078 [website]

Though pitched at a cell flow cytometry audience, this is a useful manuscript for understanding flow cytometry nomenclautre and the importance of characterizing fluorescence sensitivity.
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 [website]

2015

Thorough review on EV flow cytometry pitched at a beginner level.
Nolan J.P., Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects, Current Protocols in Cytometry, 2015, DOI: 10.1002/0471142956.cy1314s73 [website]

First paper demonstrating EV diameter derivation from membrane intercalating dye and first EV paper using fluorescence calibration.
Stoner S.A., Duggan E., Condello D., Guerrero A., Turk J.R., Narayanan P. K., Nolan J.P. 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 [website]

2014

Paper demonstrating the cross-platform sensitivity and resolution differences when analyzing EVs.
van der Pol E., Coumans F. A. W., Grootemaat A. E., Gardiner C., Sargent I. L., Harrison P., Sturk A., van Leeuwen T. G. , Nieuwland R., 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 [website]

Standardization study demonstrating the utility of Q&b for cell analysis.
Perfetto, S. P., Chattopadhyay, P. K., Wood, J. , Nguyen, R. , Ambrozak, D. , Hill, J. P. and Roederer, M., 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 [website]

2012

First paper to demonstrate swarm detection and perform EV light scatter calibration.
van der Pol E., van Gemert M. J. C., Sturk A., Nieuwland R., T. G. Van Leeuwen 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 [website]

2008

ISAC position paper outlining the minimum requirements for reporting a flow cytometry experiemnt. While predominantly developed for cellular analysis many of the criteria are applicable to small particle analysis. This framework was tailored to small particle analysis in the MIFlowCyt-EV framework, 2020.
Lee JA, Spidlen J, Boyce K, Cai J, Crosbie N, Dalphin M, Furlong J, Gasparetto M, Goldberg M, Goralczyk EM, Hyun B, Jansen K, Kollmann T, Kong M, Leif R, McWeeney S, Moloshok TD, Moore W, Nolan G, Nolan J, Nikolich-Zugich J, Parrish D, Purcell B, Qian Y, Selvaraj B, Smith C, Tchuvatkina O, Wertheimer A, Wilkinson P, Wilson C, Wood J, Zigon R; International Society for Advancement of Cytometry Data Standards Task Force, Scheuermann RH, Brinkman RR. MIFlowCyt: the minimum information about a Flow Cytometry Experiment. Cytometry A. 2008 Oct;73(10):926-30. doi: 10.1002/cyto.a.20623. [website]

2005

Paper outlining how MESF values are assigned.
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 [website]