Pkh26 kit

Exos were resuspended in diluent C (1 mL) and stained by PKH26 dye (4 μL) for 4 min based on the manuals of a PKH26 kit (Sigma-Aldrich, St. Louis, MO, USA). The staining procedure was terminated by dark incubation with 1% BSA (2 mL) for 1 min. The labeled exos underwent a 70-min centrifugation (110,000g) and PBS resuspension, subsequent to co-culture with NP cells for 12 h. After that, the cultured cells were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich), washed with PBS, stained by DAPI (Sigma-Aldrich), and observed using a laser scanning confocal microscope (Olympus, Tokyo, Japan).

BMDM and RAW‐derived sEVs (∼10⁸ particle/ml or concentration 5 μg/ml) were labelled using the PKH26 kit (Sigma‐Aldrich) after the first ultracentrifugation step at 100k × g. Briefly, sEV pellet from the first ultracentrifugation step was resuspended in 80 μl of Diluent C, then 93 μl of mixed PKH26/Diluent C (3 μl in 200 μl Diluent C) was added and incubated 3 min at RT in the dark. The same procedure was carried out for the ‘dye control’ where the supernatant of the first ultracentrifugation step was used. Next, 7 ml of cold filtered PBS was added to stop the labelling process and followed by ultracentrifugation at 100k × g for 80 min. The labelled sEV pellet and dye control were resuspended in PBS and used for in vitro uptake experiments. Labelled sEVs were added to the culture medium of BMDMs and BMDCs (5 × 10⁴ cell/well) with a concentration of 2 × 10³ particles per recipient cell.

PKH-26 kit (Sigma, USA, PKH26GL-1KT) was used to label exosomes according to the manufacturer’s protocol. The microtia chondrocytes were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (Beyotime, China, C1031) according to the manufacturer’s protocol. The labelled exosomes were added into the culture dish or mixed together with Gelma hydrogel and microtia chondrocytes for 24 h at the same culture dish or hydrogel. The details of labelling could be seen at Additional file 1: 1.3. We used the confocal microscopy imaging to observe the uptake of exosomes for the 2D or 3D culture.

Purified EVs were labeled with a PKH26 kit (red; Sigma–Aldrich) for labeling and uptake studies according to the manufacture’s protocols with some modifications (Zhang et al., 2018 (link)). In brief, 250 μl of purified OECs-EVs diluted in PBS mixed with 250 μl of Diluent C. In parallel, 2 μl of PKH26 dye was added to 500 μl of Diluent C for a final PKH26 concentration of 1 × 10⁻⁶ M, then the mixture was incubated with EVs solution above for 4–6 min at room temperature. Excess dye from the unlabeled EVs was neutralized with 1 ml of 5% BSA and removed by ultracentrifugation. After incubation, the EVs were resuspended in sterile PBS and centrifuged at 100,000 g for 70 min twice. The acquired PKH26-labeled EVs pellet was carefully resuspended in 100 μl of PBS. A mixture without EVs was used as a negative control to examine any carryover of PKH26 dye. For negative control, labeling was performed as described but without EVs. PKH26-labeled OECs-EVs were added to DRG cultures in FBS-free medium overnight. The DRG neurons were stained for β-tubulin III (1:200, Abcam; green) and counterstained with DAPI. The internalization of PKH26-labeled OECs-EVs was observed under a fluorescence confocal microscope (A1+, Nikon, Japan).

Fresh human neutrophils were used to assess neutrophil extracellular trap (NET) formation by use of a previously described protocol based on the PKH26 kit (Sigma-Aldrich) with minor modifications (83 (link)). In brief, 37,500 neutrophils in RPMI medium were stained with a 2 μM concentration of the red fluorescent cell linker provided in the PKH26 kit before they were challenged with A. actinomycetemcomitans for 2 h. Subsequently, the infected neutrophils were stained with a 5 μM concentration of the membrane-impermeable DNA dye Sytox green (Thermo Fisher Scientific), and the samples were fixed with PFA. Finally, NETs were visualized by immunofluorescence confocal microscopy (Zeiss Celldiscoverer 7). A 0.125-μg/mL (20 nM) concentration of PMA in PRMI medium was added to neutrophils as a positive control for NETosis, and fresh RPMI medium was used as a negative control. All experiments were performed as three biological replicates with triplicate measurements per condition.

The MuVs were labelled using PKH26 kit (Sigma‐Aldrich®). Briefly, after adding 100 μL of Diluent C to the MuV suspension, 100 μL of 4 μM PKH26 solution were added to the sample. After 5 min of incubation, 1 mL PBS was added, and the MuVs were washed using a 100 K concentrators, 15 000 g at 4°C for 10 min. The MuVs were washed three times in PBS using the 100 K concentrators before being mixed with the cell media for treatment. All cell cultures treated with MuVs were compared and normalized to untreated cell cultures.