Chapter 3 80 Control type Rationale PBS Blanc - Background control PBS + mAbs mAb mediated background control PBS + Isotypes Isotype mediated background control Unstained sample Autofluorescence of unstained sample Sample + Single stain Fluorescence compensation purpose Sample + Isotype Unspecific binding of antibodies used Sample + Double stain Multiparameteric detection of sample of interest Sample + Double stain + Detergent Treatment Confirmation that detected events are of biological nature Supplementary Table 3 - Control types and the rationale for their use. Each control listed above is essential for the multiparametric detection of human plasma-derived single EV. Parameter Settings Magnification: 60x Lasers: 405nm – 488nm – 642nm - SSC (785 nm) Voltage: 120mW – 200mW – 150mW – 1.25mW Fluidics: Low Speed & High Sensitivity Autofocus: ON Remove Speedbead: Unchecked Core Widt: 6 µm (Override) Acquisition time: 180 seconds Supplementary Table 4 - Acquisition parameter settings for the multiparametric detection of single EV in human plasma samples using the ISx MKII imaging flow cytometer. Lasers were turned on as applicable for each experiment. SSC: Side Scatter.
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