Synergy him microplate reader

To investigate the cytotoxicity of HTON to three kinds of cells, cells (1 × 10⁴ cells/well) were seeded in 96-well plates and incubated with culture media containing different concentrations of HTON (12.5–200 μg/mL) for 24 h. The CCK-8 reagent (Beyotime, Shanghai, China) was added into each well followed by 60-min incubation at 37 °C, and then the absorbance at 450 nm was collected on a BioTek Synergy HIM microplate reader (CA, USA). Cytotoxicity was expressed as the percentage of cell viability compared to blank control. Similarly, the cytotoxicity of STT and AA at the concentration of 31.2–1000 μg/mL was also measured. As to the cytotoxicity of the HTON@Gel dressing, the 20 μL freshly prepared dressing was paved at the bottom of a 96-well plate, and then incubated in the cell incubator for 10 min for natural gelling followed by 24-h cell culture (10⁴ cells/well) and CCK-8 test.
For cellular proliferation, 2 × 10³ cells per well were seeded in 96-well plates and incubated with high-glucose culture medium containing 200 μg/mL HTON for 7 days. In the HTON + VIS + STT and HTON + VIS + AA groups, 250 μg/mL STT and AA were individually added with HTON solution. For VIS-involved groups, cells were irradiated under the Xe lamp (0.1 W/cm²) for 3 min every day and CCK-8 was used to detect cell viability at different time points (0, 1, 3, 5, and 7 days).

Agilent 1200 high performance liquid chromatography (Agilent Technologies, Inc., Beijing, China), Bruker Avance III 400 HD NMR spectrometer (Bruker, Beijing, China) and Bruker Autoflex MALDI-TOF mass spectrometer (Bruker, Beijing, China) were used to purify and identify the target product, respectively. Bruker TENSOR-27 Infrared Spectroscope (Bruker, Beijing, China), UV-2550 UV–Vis spectrophotometer (Shimadzu Company, Kyoto, Japan) and IONTOF time of flight secondary ion mass spectrometer (ToF-SIMS V) (IONTOF, Munster, Germany) were used to characterize functionalized magnetic nanoprobes. Quantitative mass spectrometry analysis of proteins was performed on a Waters Xevo-G2-Q-TOF spectrometer (Waters, Milford, MA, USA), which was coupled to a Dionex nano-LC3500 System (Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China). SYNERGY HIM Microplate reader (Bio Tek, Winowski, VT, USA) was used to measure the biological activity of baicalin and modified baicalin.

Cells were seeded into 96-well plates (1 × 10³ cells/well) 24 h after transfection. Plates were incubated in a humid incubator at 37 °C for 1, 2, 3, 4, 5, and 6 days. Then 20 μL MTT at a concentration of 5 mg/ml in phosphate-buffered saline (PBS) was added into each well. The plates were incubated at 37 °C for a further 4 h, after which we removed the medium and added 150 μL dimethyl sulfoxide (DMSO) to each well. The absorbance was read at 490 nm by the Synergy HIM microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).