FCMPASS software aids extracellular vesicle light scatter standardization’
FCMPASS software aids extracellular vesicle light scatter standardization’
The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analysing EVs due to it being a high-throughput, multi-parametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardisation exists for EV flow cytometry which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard-to-interpret, compare,and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardisation, utilising flow cytometer post-acquisition analysis software (FCMPASS). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter-diameter curve and scatter-refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardised units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.
Welsh, Joshua
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Horak, Peter
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Wilkinson, James
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Ford, Verity J.
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Jones, Jennifer C.
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Smith, David
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Holloway, Judith
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Englyst, Nicola
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Welsh, Joshua
3bbcb204-ddcb-49c9-8cd4-f5e2906a666e
Horak, Peter
520489b5-ccc7-4d29-bb30-c1e36436ea03
Wilkinson, James
73483cf3-d9f2-4688-9b09-1c84257884ca
Ford, Verity J.
8c5e698a-08b5-472d-b3c0-434be2dcf91c
Jones, Jennifer C.
f40f2dec-9a7a-4c95-8b6a-e6fb7cf5f5ca
Smith, David
217b19f1-7588-4d54-a062-8394eea6dea1
Holloway, Judith
f22f45f3-6fc8-4a4c-bc6c-24add507037c
Englyst, Nicola
f84399af-7265-4224-b556-102c3aa272b0
Welsh, Joshua, Horak, Peter, Wilkinson, James, Ford, Verity J., Jones, Jennifer C., Smith, David, Holloway, Judith and Englyst, Nicola
(2019)
FCMPASS software aids extracellular vesicle light scatter standardization’.
Cytometry Part A.
(doi:10.1002/cyto.a.23782).
Abstract
The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analysing EVs due to it being a high-throughput, multi-parametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardisation exists for EV flow cytometry which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard-to-interpret, compare,and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardisation, utilising flow cytometer post-acquisition analysis software (FCMPASS). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter-diameter curve and scatter-refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardised units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.
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FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization
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Submitted date: 13 December 2018
Accepted/In Press date: 4 April 2019
e-pub ahead of print date: 28 June 2019
Identifiers
Local EPrints ID: 430812
URI: http://eprints.soton.ac.uk/id/eprint/430812
ISSN: 1552-4922
PURE UUID: a11bbc89-f153-4375-b60d-11c08a5d6fe8
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Date deposited: 14 May 2019 16:30
Last modified: 16 Mar 2024 07:50
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Author:
Joshua Welsh
Author:
Peter Horak
Author:
Verity J. Ford
Author:
Jennifer C. Jones
Author:
David Smith
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