2.8. Exosome isolation, labeling, and treatment
FBS contained in the complete DMEM was replaced with exosome-depleted
FBS (Gibco, USA, A2720803) to isolate cell-derived exosomes without
bovine exosome. Then, Caco-2 cells at 70-90% confluency in flask were
cultured with this exosome-free medium. Three days later, the media were
collected from the flasks to isolate exosomes. Exosomes were isolated
using Total Exosome Isolation Reagent (Invitrogen, USA, 4478359)
following the user manual. The isolated exosomes were suspended in PBS
and fluorescence-labeled by 18:1 PE-TopFluor® AF488 (Avanti® Polar
Lipid, UAA, 810386C). Before the labeling, exosomes in PBS were counted
using EXOCET Exosome Quantitation Kit (System Biosciences, USA,
EXOCET96A-1) following the user manual. Exosomes at
4×108 particles/ml were directly treated into the
mono-cultured BBB for 2 h. Exosomes at 8×108particles/ml were treated into the gut barrier module of GBA chip for 6
h.