Avoiding Artifacts in EV samples:
Part 1 – the Use of Tween®

Many different methods have been developed for preparation of EVs, before characterization. Along with generally accepted methods are some “tricks of the trade” such as the addition of Tween^® or BSA; however, some of those additions are problematic, in that they create significant background particles.

The use of Tween^® in low concentrations for EV sample preparation has been recommended by several vendors. The creation of background particles may come as a surprise to some researchers, where filtration before addition to a sample may not entirely solve the issue for a couple of reasons. First, many syringe filters shed particles of their own and require conditioning before filtration of any diluent; therefore, one must be careful not to compound the problem when taking this step. Second, as Tween^® is essentially detergent, it is likely that the interaction of Tween^® with the protein in EV samples will generate additional, measurable particles after a filtration step. We present here some simple tests to illustrate the problem.

Figure 1 shows Nanoparticle Tracking Analysis measurement of just PBS, a null set. Only three particles were tracked when using a typical SOP for EVs, so this PBS is clean. Figures 2, 3 and 4 show the effect of adding Tween^® to PBS, even though the additive is only present in low amounts (0.1% and 0.3% concentrations). Notably, our ZetaView^® system is too sensitive at a concentration of 0.5% Tween^®, which creates so many surfactant nanoparticles that NTA analysis isn’t feasible.