An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 71 Framework Criteria What to report Please complete each criterion 3.6 Procedural controls. State whether procedural controls were included. If used, state the procedure and if the procedural controls were acquired at the same settings and during the same experiment as stained samples. No procedural controls were used as no further sample processing was performed after labelling with reagents. 3.7 Serial dilutions. State whether serial dilutions were performed on samples and note the dilution range and manner of testing. The fluorescence and/or scatter signal intensity would ideally be reported in standard units (see Section 4.3, 4.4) but arbitrary units can also be used. This data is best reported by plotting the recorded number events/ concentration over a set period of time at different sample dilution. The median fluorescence intensity at each of the dilutions should also ideally be plotted on the same or a separate plot. Serial dilution samples were measured at the same (initial) dilution and at the same concentration as matched stained controls and were recorded during the same experiment at the same imaging flow cytometer with acquisition settings similar to all other samples, including laser power and flow rate. Four times 4-fold dilution was performed by mixing 100 uL of (stained) sample with 300 uL of fPBS. Correlation analysis showed a linear correlation between the concentration of double-positive fluorescent EV (CFSE+Tetraspanin+) and dilution rate (R^2=0,93). Fluorescent intensities remained stable: ~113 ERF CFSE and ~32 ERF APC. 3.8. Detergent treated EVsamples State whether samples were detergent treated to assess lability. If utilized, state what detergent was used, the end concentration of the detergent, and what the results were of the lysis. A 10% (v/v) Triton X-100 stock solution was made by dissolving 1 mL of TritonX-100 in 9 mL of fPBS. All samples (buffer alone, buffer plus reagents, unstained samples, single-stained samples, and double-stained samples) were treated with 20 µL of the Triton X-100 stock solution (final concentration: 0.5% (v/v) per test), followed by 30 minutes of incubation at room temperature in the dark prior to acquisition. Comparison of fluorescent concentrations in the PPP samples obtained before and after detergent lysis for CFSE+, Tetraspanin+, and CFSE+Tetraspanin+ regions showed ~31%, ~64% and ~94% reduction, respectively. For CD9+CD31+ EV, a ~93% reduction was observed after detergent lysis.
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