Supplementary Figure 1: Characterization of AF4 fractions using TEM imaging and NTA analyses and examination of AF4 profiles of nanoparticles derived from cells under different culture and storage conditions.
(a) TEM analysis of particles in AF4 peaks P1 and P5 of B16-F10. The experiment was repeated independently 3 times with similar result. Scale bar, 500 nm. (b) Comparison of the hydrodynamic diameter of each fraction determined by AF4-QELS versus NTA. Individual fractions (time slice, 0.5 min/fraction) were taken every 2 min from 20 to 44 min during the AF4 time course, and subjected to NTA. Results shown are mean ± SEM (n=3 independent samples). Mode size from NTA was utilized. X-axis, time course of AF4 (min); Y-axis, hydrodynamic diameter (nm). (c) The size distribution profiles of representative fractions by NTA (input, unfractionated samples; fractions at 20, 32, and 44 min). Multiple peaks were detected for fractions at 20 and 44 min by NTA. a mode size of 126 nm of Input indicates that NTA cannot efficiently resolve polydisperse samples and is biased towards large particles. This experiment was repeated 3 times independently with similar results. (d) Particle concentration of each fraction measured by NTA. The hydrodynamic diameter of the peak fraction (28 min) was 77nm. Results shown are mean ± SEM (n=3 independent samples). (e-i) AF4 profiles of B16-F10 sEVs collected from technical (blue lines, replicate #1; red lines, replicate #2) (e) and biological replicates (red lines, QELS; blue lines, UV; black (replicate #1) and green dots (replicate #2), hydrodynamic radius; Differences in UV and QELS signal intensity is due to the different amount of input samples for two replicates) (f), kept at either 4 oC or -80 oC for one week (red lines, QELS; blue lines, UV; black (fresh) and green dots (frozen), hydrodynamic radius) (g), cells of different passage numbers (blue and red lines, UV of cells at passage 10 and 18, respectively; black dots, hydrodynamic radius) (h), and under hypoxic versus normoxic conditions for 48 h (blue and red lines, UV for samples cultured with 20% and 1% O2, respectively; black dots, hydrodynamic radius) (i). Experiments were repeated independently 3 times for (e-g) and twice for (h) with similar results. For (i), the experiment was repeated with 3 different cell lines independently with similar results. (j) AF4 and (k) TEM analysis of nanoparticles isolated in parallel from the blank media control and CM of 3-day cultures of B16-F10 and MDA-MB-4175. This experiment was done once. (Red and Blue lines, UV; black dots, hydrodynamic radius; Scale bar, 200 nm.) Statistical source data are provided in Supplementary Table 8.
