Figure 2. General workflow.

Apoplastic wash is collected by vacuum infiltrating full Arabidopsis rosettes with an isotonic buffer, washing out the infiltrated buffer using a low-speed centrifugation spin and filtering the wash to remove any large debris. The wash fluid is then processed using differential centrifugation to isolate EVs, which can be stained with the lipophilic dye DiOC₆. After the EVs are washed and pelleted again to remove excess dye, DiOC₆ fluorescence in the EV pellet can be measured to indirectly quantify the amount of EVs.