fig2

Figure 2. Analysis of six sample dilutions from non-fluorescent SiNPs across platforms. Dot plots displaying SSC-H vs. FITC-H or FSC-H from the total number of events of buffer control (left panel) and non-fluorescent SiNPs measured for 2 min (A) on the NF with an SSC (488 nm) threshold, (B) on the IF with an rw-FSC (488 nm) threshold, and (C) on the CF with a VSSC (405 nm) threshold. First, events were gated to minimize the inclusion of aggregates and doublets after inspection of area, height, or width parameters [Supplementary Figure 1]. Second, the indicated gate (red) was used to select the 113 nm SiNP population across dilutions and instruments. Note that the CF SiNP 1:100 and 1:500 samples were too concentrated to adequately gate around this population (red arrows). Samples were diluted as indicated in the top row (from 1:100, 1:500, 1:1,000, 1:2,000, 1:4,000, and 1:8,000, left to right); (D-F) Concentration of detected events across dilutions was calculated for each 113 nm gated sample following normalization for each instrument flow rate; (G-I) Median scatter intensity values (a.u.) from the gated 113 nm population were plotted for the NF, IF, and CF, respectively, for each diluted sample. The blue box indicates the samples that followed the expected event reduction trend while keeping a constant scattering intensity for this population. The figure shows data obtained from 6 measured samples originating from the same stock solution. NF: NanoFCM; F: BD Influx; CF: CytoFLEX LX; FITC: fluorescein isothiocyanate; FSC: forward scatter; FSC-H: forward scatter height; rw-FSC: reduced wide-angle forward scatter; SSC: side scatter; SSC-H: side scatter height; VSSC: violet side scatter; VSSC-H: violet side scatter height; SiNPs: silica nanoparticles; a.u.: arbitrary units.