Mk3 microplate reader

The sera of all participants were tested for the specific IgG to T. gondii using commercial ELISA Kit for IgG Antibody to Toxoplasma (Haitai Biomed, Zhuhai, China), according to manufacturer’s instruction. Briefly, the serum sample was diluted to a ratio of 1:100, followed by adding to test well in the antigen-coated plate and incubated at 37 °C for 30 minutes (min). After intensive washing for three times with washing solution, 50 μL peroxidase-conjugated anti-human IgG was added to the wells. After 30 min incubation at 37 °C, each well was washed with washing solution for three times. Then, 50 μL “A” solution and 50 μL “B” solution were added to test wells, and the plate was incubated at 37 °C. Ten min later, reaction was stopped by adding 50 μL stopping solution. Microplates were read at an optical density (OD) of 450 nm in the MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) and ratios (OD 450 value of serum sample/OD 450 value of negative control) were calculated after correction for the OD 450 value of the blank well. The serum samples were considered positive when the ratio was ≥2.1.

HSS was kindly provided by Professor Sun Xuejun of the Secondary Military Medical University, ROC. Solutions were freshly prepared every week and maintained at 4 °C to ensure a stable hydrogen concentration of 0.6 mM. High-purity OA was purchased from Sigma (St. Louis, MO, USA). MDA and MPO assay kits were obtained from the Nanjing Jiancheng Bioengineering Institute, China. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were provided by Peprotech (NJ, USA). Equipment in this study included MK3 microplate reader (Thermo-Scientific, MC, USA), MK2 microplate washer (Thermo-Scientific, MC, USA), electro-heating standing-temperature cultivator (Shanghai Yuejin Medical Instruments Factory, China), TGL-168 centrifuge (Shanghai Anting Scientific Instrument Factory, China), SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA, USA), micro-electric tissue homogenizer (Kimble, USA), GM3000 blood gas analyzer (Instrumentation Laboratory, MA, USA), and light microscopy (Olympus, Japan).

Cells were grown in 96-well plates (3×10³ cells/well) for 24 h and then incubated with either micelles (vector: DOX = 10:1(W/W)) or free DOX at equivalent concentrations for 72 h. Then, 10 μL of CCK-8 solution (Beyotime, CA) was added to each well. The cells were further incubated at 37°C for approximately 45 min. The absorbance was measured at 450 nm using an MK-3 microplate reader (Thermo Fisher Scientific, United States). The cell apoptosis rate was calculated according to the following formula: apoptosis rate = [(OD₄₅₀ control well-OD₄₅₀ administration well)/OD₄₅₀ control well]×100%. The measurements were repeated three times, and the absorbance value of the untreated control samples was considered as 100% viability.

Enhanced Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Beijing, China) was applied to examine cell proliferation. The cells in logarithmic growth phase were digested with trypsin, washed by phosphate-buffered saline (PBS), and suspended in the culture medium. Then, 2,000 cells in 100 μl medium were added into one pore of 96-well plates, 10 μl enhanced CCK-8 solution was added, and incubated for 1 h. The value of optical density was detected with the help of an MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at the wavelength of 450 nm.

A total number of 5 × 10⁴ HT29 cells were seeded in the wells of a 24-well plate and routinely cultured for 24 h. Then, with the blank culture solution as the control, 5 mg of sterilized PVA, PVA/DOX, PVA/MoS₂, or PVA/MoS₂/DOX microspheres was added into the wells of a 24-well plate (containing 2.5 mL DMEM per well). The cells were then irradiated with NIR laser (808 nm, 1.0 W/cm², 5 min), the metabolic activity of HT29 cells was measured by Dead/Live kit and CCK-8 kit, using an MK3 micro-plate reader (Thermo Fisher, Waltham, MA, USA) and Olympus BX43 fluorescence microscope, respectively.