An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 41 Fluorescent calibration for standardized reporting As mentioned before, flow cytometers differ in their fluorescent sensitivity and dynamic range, and therefore data comparison between different instruments is hindered. In order to improve data comparison fluorescent calibration must be performed to convert arbitrary units (a.u.) into standardized units. To this end, we used commercially available Rainbow Calibration Particles (RCP) with known reference values in terms of the Equivalent number of Reference Fluorophores (ERF). Using the same settings as applied for EV measurements, we measured the Mean Fluorescent Intensity (MFI) of each of the four RCP bead populations (1 blanc – 3 fluorescent) generated for each channel used in our setup (Figure 4a). Using the blank bead populations, we established the lower detection thresholds for fluorescent detection channels Ch02 (CFSE) and Ch05 (APC). We then calculated the respective logarithmic values of each peak (Figure 4b), and performed a linear regression analysis of the ERF values against the MFI for peaks 2 to 4, omitting the blanc beads as these represent PS beads without fluorophores (Figure 4c). In the example of the double-stained PPP sample presented in Figure 3b without fluorescent calibration, we next converted the measured fluorescent intensities for CFSE and APC of each event into their respective ERF values (Figure 4d). Lower fluorescent thresholds were converted accordingly and resulted in 35.40 and 6.40 ERF for CFSE and APC, respectively. Upper fluorescent thresholds were calculated at 3776 and 123 ERF for CFSE and APC, respectively. For the double-positive fluorescent population, this conversion resulted in median values of 138.09 ERF CFSE and 27.88 ERF APC. These data show that the fluorescent intensities generated by imaging flow cytometry can be readily converted into standardized units, which, in turn, enhances the comparability of the generated data with other instruments using the same filter sets.
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