Chapter 3 42 Figure 4 – Fluorescent calibration allows reporting of fluorescent intensities in standardized units. a) The median fluorescent intensities (MFI) of each peak of FITC and APC ERF (Equivalent number of Reference Fluorophores) calibration beads was measured with the same instrument/acquisition settings applied as used for EV acquisition. b) Calculation of the log of the MFI and ERF values (provided by the bead manufacturer). c) For each of the used detection channels, the log of the MFI corresponding to the fluorescent peaks (P2-P4) was plotted on the x-axis, and the log of the ERF values on the y-axis; linear regression analysis was performed. d) Representative example of uncalibrated data (left) and corresponding ERF calibrated data (right). Testing EV coincidence occurrence through serial dilution The detection of multiple EV as a single event can lead to false interpretation of the data (e.g. underestimation of the concentration of particles of interest). To examine the accuracy of quantification of EV from PPP by our IFCM protocol, we double stained the 5 PPP samples with CFDA-SE and the anti-tetraspanin antibody mixture and performed a serial dilution experiment. The concentrations and ERF of double-positive particles in each PPP sample obtained after four 4-fold dilution steps were analyzed using a linear regression model, with the results shown in Figure 5. All data shown were used in the analysis and R2 calculation.
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