4.3.4. XenoLight DiR Labelling

To detect EV uptake by cells, the undiluted EVs-containing suspension was incubated with 2 µM of XenoLight DiR Fluorescent Dye (PerkinElmer, Waltham, MA, USA) diluted in diluent C (Sigma-Aldrich, Darmstadt, Germany) for 30 min. The resulting solution was then washed twice in sterile PBS. hTCEpi cells were cultured with labelled-HCjE-Gi-derived EVs diluted in conjunctival KSFM (BPE-free), and HCjE-Gi cells were cultured with labelled-hTCEpi-derived EVs diluted in corneal KSFM (BPE-free). As a control, HCjE-Gi cells were cultured with their own unlabeled-derived EVs in conjunctival KSFM (BPE-free) and hTCEpi cells with their own unlabeled-derived EVs in corneal KSFM (BPE-free). This final suspension was analyzed using a BD Accuri C6 flow cytometer (Laser 640 nm, filter 780/60, BD Biosciences, Franklin Lakes, NJ, USA) and the results analyzed using BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ, USA).