Information about figures for Reliable measurements of extracellular vesicles by clinical flow cytometry


Figure 2: Figure 2. (A) The scattering intensities of hollow organosilica beads (HOBs) with a diameter of 180 nm,
silica beads with a diameter of 183 nm and polystyrene beads with a diameter of 203 nm show that beads
with higher refractive indices have a higher light scattering intensity. Polystyrene and silica beads have a
refractive index of 1.633 and 1.475, respectively. Because the core of HOBs contains water, HOBs scatter
light less efficiently than both polystyrene and silica. (B) Side scattering intensity versus diameter
measured (symbols) by flow cytometry (A60-Micro, Apogee Flow Systems, UK) and calculated (lines)
with Mie theory for polystyrene beads (squares), silica beads (circles), HOBs (triangles) and EVs (shaded
area between dotted lines). Please note that the vertical scale is logarithmic. Mie theory calculations are
performed with Rosetta Calibration software (Exometry, The Netherlands) assuming a refractive index of
1.633 for polystyrene beads and 1.475 for silica beads. HOBs are modelled as core-shell particles, with a
core refractive index of 1.343, a shell refractive index of 1.475, and a shell thickness of 10 nm. EVs were
also modelled as core-shell particles, but with a core refractive index ranging from 1.343 to 1.36, a shell
refractive index of 1.46, and a shell thickness of 5 nm. The side scattering intensity increases with
increasing diameter, but also with increasing refractive index. Hence, for this flow cytometer, polystyrene
beads of 200 nm scatter light more efficiently than 1,000 nm EVs (arrow).

Figure 2a
Histograms of data from HOBs, silica beads and polystyrene beads were made using the MATLAB script "CSV_reader_hist_scatter.m" 
in MATLAB version 2010b. The data itself can be found in "LALS_203nm_PSbeads.csv" (PS beads), "LALS_182nm_silicabeads.csv" 
(silica beads) and "LALS_180nm_HOBs.csv" (HOBs). The data comes from beads measured on the A60-Micro from Apogee Flow Systems, 
measured at 3.01 microL/min. 

Figure 2b
Information about the settings of the flow cytometer and dilutions for the measurements can be found in: 
20160622_EvdP_Hollow_silica_beads_Zoltan.xlsx

Data from the beads:
The polystyrene beads measured are (from small to large):
"10_PS_100nm_dil1E6.fcs"  
"11_PS_125nm_dil1E6.fcs"
"12_PS_150nm_dil1E5.fcs"
"13_PS_203nm_dil1E5.fcs"
"14_PS_300nm_dil1E5.fcs"

The silica beads measured are (from small to large):
"15_Si_75nm_dil1E6.fcs"
"02_Si_182nm_dil1E5.fcs"
"04_Si_315nm_dil1E5.fcs"
"05_Si_321nm_dil1E5.fcs"
"06_Si_405nm_dil1E4.fcs"
"07_Si_548nm_dil1E4.fcs"

The HOBs measured are (from small to large):
"19_HS3_180_nm_hollow_silica_beads_dil2E4.fcs"
"16_HS5_361_nm_hollow_silica_beads_dil2E3.fcs"


Figure 3: The side scattering intensities and fluorescence intensities of human plasma labeled with cluster
of differentiation (CD) 61 conjugated to allophycocyanin (APC) can be used to determine the populations
of CD61+ extracellular vesicles (EVs), of CD61- particles, residual platelets, and aggregates originating
from the fluorescent reagent. The results are (A) uncalibrated, thus in arbitrary units, and (B) calibrated
with the diameter of the EVs in nm and the fluorescence intensity in molecules of equivalent soluble
fluorophore (MESF). Without the red outlined regions, uncalibrated results would be difficult to interpret,
whereas the calibrated results are insightful and comparable to other data of platelet-derived EVs.

The measured sample was from a healthy volunteer as part of the following study:
https://www.aleksandragasecka.com/publications/manuscripts/Gasecka_2019_Ticagrelor_attenuates_EV_increase_after_AMI.pdf  

The .fcs file containing the flow cytometry data from a CD61 labelled sample of EVs for both calibrated and uncalibrated figures:  
"01A 40x CD61-APC CD62p-PE_A1_0_PSD_settings_1_PSD_settings_2_Flow-SR_medium_PBS_MESF_PSD_1.fcs"

The sample is measured on the A-60 Micro flow cytometer from Apogee Flow Systems, where the sample was analysed for 
2 minutes at 3.01 microL/min. More information about the sample preparation can be found in: 
https://onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1111%2Fjth.14689&file=jth14689-sup-0001-SupInfo1.pdf 

Dataset for Rosetta Calibration: 
The .fcs file containing the flow cytometry data from the Exometry beads used for calibrating the EV data: 
"d03_20180819AG_Exo beads_3_01_F3_0.fcs"

The file was calibrated using the Falcon software from Exometry B.V. Falcon uses the data from the calibration beads and data from 
Mie theory to enable conversion between arbitrary units and defined units such as diameter in nanometer.
Information about the settings of the flow cytometer and the settings used to define the gates in Falcon can be found in:
"Define_gates.xlsx"
"Results_gates.xlsx"
"Settings_PSD_1.png"