Dm il led inverted phase contrast microscope

In this work, 0.2 mol/L of H₂O₂ was added in DMEM, and the pH of solutions was adjusted to 6.4 with HCl. After that, 8,000 cells/well of HT29 cells were cultured with 0.1mL DMEM in 96-well-plate for 12 h. Then, FMPBs in fresh DMEM (0, 0.25, 0.5, and 1 mg/mL) were added in 96-well-plates (n = 3). The solution was then irradiated with 808-nm laser (1 W/cm²) for 5 min. Finally, the treated cells were incubated for 24 h. CCK-8 kit and dead/live kit were used to study the cell viability according to the instructions. Leica DM IL LED inverted phase-contrast microscope was used to capture the stained cells (live cells showed green fluorescence and dead cells with red).

An MTT assay was applied to measure the in vitro cytotoxicity of the {(Au⁰)₁₀₀G5.NH₂-FI-DOTA(Mn)-HA} NPs in a routine culture environment with 10% FBS (heat-inactivated, 1% penicillin-streptomycin) and 5% CO₂ at 37 °C.
HCCLM3 cells suspended in medium were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well with 200 μL per well and were incubated overnight. NPs with an Mn concentration ranging from 0–100 μg/mL were added into each well, and the cells were incubated for an additional 24 h. The mixture was then carefully removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 20 μL of MTT solution (5 mg/mL in PBS) was added into each well, and the cells were cultured for another 4 h at 37 °C and 5% CO₂. To dissolve the insoluble formazan crystals, the medium was carefully discarded and replaced with 200 μL of DMSO. Finally, the absorbance of each sample was measured at 570 nm using a Thermo Fisher Scientific Multiskan MK3 ELISA reader (Thermo Fisher Scientific, Hudson, NH).
The morphology of the HCCLM3 cells was observed to evaluate the cytotoxicity of the {(Au⁰)₁₀₀G5.NH₂-FI-DOTA(Mn)-HA} NPs after the cells were treated with the NPs at Mn concentrations of 0, 10, 20, 50, 75, and 100 μg/mL for 24 h. A Leica DM IL LED inverted phase-contrast microscope was employed to observe the cell morphology of each sample at a magnification of 200×.

HeLa cells were purchased from the Institute of Biochemistry and Cell Biology (the Chinese Academy of Sciences, Shanghai, China) and cultured in DMEM containing 5% FBS and 1% antibiotics at 37°C and 5% CO₂.
HeLa cells (10,000 cells/well) were seeded in 96-well plates overnight. The Fe₃O₄@HA nanoparticles at various Fe concentrations (0.025, 0.05, 0.1, 0.2, and 0.4 mM) were next incubated with the HeLa cells for another 24 h in 200 μL of DMEM. HeLa cells treated with PBS were used as a control. The morphology of HeLa cells was further observed by phase contrast microscopy (Leica DM IL LED inverted phase contrast microscope) at a magnification of 200 times.
In vitro cytotoxicity was further quantitively confirmed by the MTT assay. Similar to the protocols described earlier, HeLa cells (10,000 cells/well) were seeded in 96-well plates overnight. The Fe₃O₄@HA nanoparticles at various Fe concentrations (0.025, 0.05, 0.1, 0.2, and 0.4 mM) were then incubated with the HeLa cells for another 24 h in 200 μL DMEM. Furthermore, the cells were rinsed three times with PBS and then incubated in 100 μL of FBS-free DMEM medium containing 10% MTT for 4 h. After removal of the medium, the MTT assay was performed according to the manufacturer’s instructions. For each concentration, three parallel wells were measured to give the average values and standard deviations.