Exosparkler exosome membrane labeling kit deep red

AML12 and LX-2 cells were seeded at a density of 3 × 10⁴ cells/well in 6-well plates. AML12 cells were treated with serum-free medium for 4 days for differentiation into hepatocytes. The LX-2 cells were treated with TGF-β1 for 48 h for activation. EVs were labeled with the ExoSparkler Exosome Membrane Labeling Kit-Deep Red (Dojindo, Kumamoto, Japan, #EX03). The cells were washed twice with PBS and then loaded with EVs with an equal amount of protein (3 ng proteins/mL) for 4 h at 37 °C with 95% air-5% CO₂. After 4 h, the cells were observed using an all-in-one fluorescence microscope BZ-X810 (Keyence, Osaka, Japan). The medium was discarded, and the cells were collected by 0.025% trypsin and fixed with 10% paraformaldehyde in PBS for 15 min. The intracellular uptake of EVs was measured by flow cytometry (FACS Celesta multi-color cell analyzer, Becton Dickinson, Franklin Lakes, NJ, USA) as in our previous study [19 (link),20 (link)]. The deep red fluorescence intensity of the cells was analyzed using flow cytometry. The transfected cells were detected by subtracting the autofluorescence. Cells were analyzed by acquiring 10,000 events and the intracellular uptake efficiency was calculated based on the percentage (%) of deep-red positive cells among all collected cells.