Pkh26 red fluorescent cell linker midi kit

The P/T or P/T/I scaffolds were trimmed into desired cylinders (size of 2 mm in diameter and 4 mm in length) and then immersed in HFS solution in glassware with a vacuum valve, and then the air within the scaffolds was removed by vacuum followed by the HFS solution penetration. The amount of HFS loaded into the scaffold was 466.7 ± 9.4 µg protein per cylinder. HFS were labelled with PKH26 Red Fluorescent Cell Linker Midi Kit (Sigma-Aldrich, Burlington, MA, USA) according to the manufacturer’s protocol. More specifically, PKH26 dye was diluted in Diluent C containing HFS solution. Excess dye was quenched by 10% BSA (Sigma, Burlington, MA, USA) in PBS. After adding 0.971 M sucrose solution, the solution was centrifuged at 190,000× g for 2 h at 4 °C. The pellet was resuspended and transferred to Amicon 10 kDa MWCO (Millipore, Burlington, MA, USA), and then spun at 3000× g for 40 min. The concentrate from the Amicon column was PKH26-labelled HFS and showed red fluorescence (ex/em ~551 nm/~567 nm).

For EV-uptake experiments, purified EVs were fluorescently labelled using PKH26 Red Fluorescent Cell Linker Midi Kit (Sigma-Aldrich). EVs isolated by ultracentrifugation and resuspended in 1 mL of PBS were labelled with the PKH26 dye (2 µM final concentration) according to the manufacturer’s instructions.