fig4
Figure 4. Quantitative analysis of fluorescent rEVs. (A-C) Linear regression analysis showing concentration of measured particles across three selected dilutions (1:1,000, 1:2,000, 1:4,000 for NF and 1:8,000, 1:16,000, and 1:32,000 for IF and CF). R2 values are indicated in each graph to show goodness-of-fit. Detected events in each sample were normalized by buffer control and flow rate according to detection strategy and each instrument (SSCt, SSCt + FLg, or FLt); (D-F) Median fluorescence intensity of gated fluorescent rEVs across three selected dilutions and different detection strategies (SSCt + FLg or FLt); (G) Bar graph of measured rEV average concentrations using the indicated three optimal dilutions across different threshold options (SSCt or FLt). For comparison, we used a FLg over the total events detected with SSCt to select for the fluorescent rEV population on the NF, IF, and CF. Concentration measurements are corrected for time, dilution factor, PBS buffer control, and flow rate for each instrument and detection mode. rSD is calculated from the selected three dilutions performed from the same sample for each detection strategy (SSCt, SSCt + FLg, or FLt). The data shown is representative of two independently performed experiments acquiring six diluted samples from the same stock on each instrument. CF: CytoFLEX LX; FL: fluorescence; FLg: fluorescence gate; FLt: fluorescence threshold; IF: BD Influx; NF: NanoFCM; PBS: phosphate-buffered saline; rEVs: recombinant extracellular vesicles; rSD: robust standard deviation; SSCt: side scatter threshold.
