Micro reader

The fluorescence imaging and Perls’ staining (neutral red method) were adopted to observe the cellular uptake of the IMD. First, 4T1 cells (2 × 10⁵ cells/well) were seeded in a confocal dish or 6-well plates and cultured overnight in RPMI-1640 medium at 37°C with 5% CO₂. Then, 4T1 cells were incubated with PBS, IMD, or IMD with magnetic targeting (containing DOX 20 μg/ml) at 37°C (5% CO₂) for 6 h. Alternatively, IMD at different concentrations ([IO]: 0, 0.2, 0.4 mg/ml) were added to cells and incubated for 6 h. Finally, the cells were stained with DAPI or Prussian blue/neutral red. An inverted microscope (XDS-3, Optika, Italy) or confocal microscope (TCS SP8, Leica, Germany) were used to obtain the data.
The cytotoxicity of DOX, IM, and IMD was investigated using an MTT assay. Briefly, 4T1 cells were transferred to 96-well plates with a density of 8 × 10³ cells/well and incubated overnight. Afterwards, DOX, IM, or IMD (100 μL) of different concentrations were added to each well. After incubation for 12 h or 24 h, the drug-containing medium was discarded, replaced with RPMI-1640 containing MTT (10 μL; 5 mg/ml), and further incubated for 4 h. Finally, the media was removed, and DMSO (100 μL) was added to each well. The absorbance was measured under a micro reader (Spark, Tecan, Switzerland) at 570 nm.

To measure lysyl oxidase (LOX) activity, supernatants were obtained 7 days after the induction of MSCs to OBs. The LOX assay reaction solution is a mixture of 20 μl Amplite™ HRP substrate stock solution (Abcam), 20 μl HRP (50 U/ml, Abcam), and 5 ml assay buffer (Abcam). A total of 50 μl supernatant and 50 μl LOX assay reaction solution were added to 96 wells. After incubation at 37 °C for 20 min in the dark, fluorescence was measured at Ex/Em = 540/590 using a microreader (Tecan, Maennedorf, Switzerland).