An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 69 Framework Criteria What to report Please complete each criterion 3.1 Buffer alone controls. State whether a buffer-only control was analyzed at the same settings and during the same experiment as the samples of interest. If utilized it is recommended that all samples be recorded for a consistent set period of time e.g. 5 minutes, rather than stopping analysis at a set recorded event count e.g. 100,000 events. This allows comparisons of total particle counts between controls and samples. Buffer-only control of 0.22 µm-filtered PBS (fPBS) was 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. All samples were recorded for 3 minutes to allow comparisons of total particle counts between controls and samples. In gerneral, <10 fluorescent events were acquired within this time period for each of the established gating regions. 3.2 Buffer with reagent controls. State whether a buffer with reagent control was analyzed at the same settings, same concentrations, and during the same experiment as the samples of interest. If used state what the results were. Buffer with reagent controls (single-stained with 12.5 µL anti-CD9 – 2.5 ng/test, 12.5 µL anti-CD63 – 83 ng/test, 12.5 µL anti-CD81 – 83 ng/test, 5 µL anti-CD31 (anti-human) – 1 ng/test, 5 µL anti-CD31 (anti-mouse) – 40 ng/ test, 100 µL of the 50 µM CFDA-SE stock solution) 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. All samples were recorded for 3 minutes to allow comparisons of total particle counts between controls and samples. In general, after 3 minutes, 600-700 fluorescent events (-APC) were recorded in buffer-control with anti-tetraspanin cocktail, <10 events in buffercontrol with anti-CD9, ~100-200 events in buffer-control with anti-CD63, ~400500 events in buffer-control with anti-CD81, ~<50 events in buffer-control with anti-mouse anti-CD31, ~200 events in buffer-control with anti-human anti-CD31 (-BV421), and <10 events in buffer-controls with CFDA-SE (CFSE) 3.3 Unstained controls. State whether unstained control samples were analyzed at the same settings and during the same experiment as stained samples. If used, state what the results were, preferably in standard units. Unstained control samples were measured at the same dilution as matched stained and isotype control samples, 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. No substantial changes in fluorescence signal were observed between unstained and matched isotype controls.
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