Pkh67 green fluorescent cell linker kit for general cell membrane labelling

Exosomes were labelled with a PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labelling (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Briefly, exosome solution in 1 mL of Diluent C was mixed with PKH67 dye solution in Diluent C and then incubated for 5 min. The reaction was stopped by adding an equal volume of 1% BSA. PKH67-labelled exosomes were isolated by ultracentrifugation and resuspended in PBS. To investigate the localisation of exosomes in AGS cells, PKH67-labelled CagA-positive exosomes or CagA-negative exosomes (5 μg/well) were incubated with AGS cells for 24 h. The cells were washed three times with PBS and then fixed in 4% paraformaldehyde. Cells were washed three times with PBS, mounted with ProLong antifade reagent (Invitrogen), and observed using a confocal laser scanning microscope LSM780 (Carl Zeiss, Germany).

NVs (20 μg) were labeled using a PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labelling (Sigma-Aldrich) as described previously^{30 (link)}. PKH67-labeled NVs were added to hepatocytes and incubated for 12 h at 37 °C in 5% CO2. The cells were washed twice with PBS after incubation, fixed with 4% formaldehyde for 15 min, and washed twice with PBS again. The cells were counterstained with DAPI before being mounted with ProLong Gold Antifade Reagents. The uptake of NVs by hepatocytes was measured via fluorescence microscopy.

Living cells within the aNFC matrix were visualised using the PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labelling (Sigma-Aldrich). Briefly, MSCs (5 × 10⁵) were labelled according to the manufacturer's instructions and embedded within a 0.1% aNFC hydrogel. For the detection of the labelled cells, the CQ1 (Yokogawa Electric Corp., Japan) high-content confocal imaging system was used.