Hoechst 33342

Dye-loaded PLGA NPs were prepared using the antisolvent nanoprecipitation method (16 (link)). Briefly, PLGA (5 mg/ml) was dissolved in DMSO as organic phase. The BODIPY and DiD dye stock solution was added into the organic solution. Then, the organic phase was injected into the aqueous phase containing 0.1% Tween-80 in PBS (1:4, v/v). Under constant stirring at the speed of 400 rpm, the PLGA NPs loaded with fluorescence probes were prepared. After that, the organic solvent was removed using an ultrafiltration device with a centrifugation speed of 4,000 rpm for 10 min. The prepared PLGA NPs were diluted in Milli-Q water, and the particle size and zeta potential were measured using a Malvern Zetasizer Nano-ZS system (Malvern Instruments, UK). The morphology of PLGA NPs was examined by transmission electron microscopy (Tecnai G2 Spirit Twin).
For in vitro testing, macrophages were incubated in a complete medium containing dye-loaded PLGA NPs for 3 h and then 2 μg/ml Hoechst 33342 (MedChemExpress) for an additional 2 min. To evaluate the effect of NP uptake on LD accumulation, macrophages were incubated in a complete medium containing PBS-loaded PLGA NPs for 3 h and then sequentially stained with 1 μg/ml BODIPY 500/510 for 3 h and 2 μg/ml Hoechst 33342 for 2 min, respectively. Then, cells were washed twice, and the medium was replaced with complete medium for live-cell imaging.

To evaluate the anti-apoptosis property of extracellular vesicles from hP-MSCs, the Hoechst 33342 (MedChem Express) was used to distinguish the apoptotic cells in the three different groups (Blank, Dox, EV). 3 × 10⁵/well hUC-MSCs were seeded into a 6-well plate. With doxorubicin treatment for 6 h and extracellular vesicle therapy for 24 h being completed, Hoechst 33342 was used to stain cells for 10 min. Under a fluorescence microscope (Nikon), the apoptotic cells were recognized as bright blue nuclei.