Thermo multiskan spectrum spectrophotometer

P727 isolate cultures were adjusted to 1 × 10⁶ CFU/mL and inoculated in 96-well plates with LB broth in the presence of 1/4 × MIC of MXF or nanoformulations. After incubation at 37 °C for 24 h, the plates were rinsed three times with 1.0 mL of PBS and dried at 25 °C. Subsequently, the adherent cells were stained with 1% crystal violet (Sigma-Aldrich, USA) for 10 min and then rinsed three times with 1.0 mL of sterile water. After the plates were dried, the dye was dissolved in 30% acetic acid, and the absorbance of the solubilized dye was detected at 590 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific Inc. MA, USA). Each treatment was assayed in three wells per plate, and the experiments were repeated three times.

The in vitro cytotoxicity of NPs against 4T1 cells was evaluated using the CCK-8 assay. 4T1 cells were seeded in 96-well plates at 1 × 10⁴ cells per well for 24 h at 37 °C in 5% CO₂. Then the cells were treated with DTX, blank NPs or DTX-loaded NPs at a concentration of 0.31, 1.25, 5.00, 20.00 or 80.00 ng/mL for 24 h or 48 h at 37 °C in 5% CO₂. After treatment, the cells were washed with PBS three times and incubated with CCK-8 solution for another 0.5 h at 37 °C in 5% CO₂. Finally, the absorbance of the cultures was measured at 450 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA). The 50% inhibitory concentration (IC₅₀) was calculated by plotting the dose–response curve using GraphPad Prism 8.0.

The in vitro cytotoxicity of DTX, CA, or nanoformulations against 4T1 cells and MDA-MB-231 cells were investigated by the CCK-8 assay. The cells were cultured in 96-well plates at 1 × 10⁴ cells per well for 24 h. Then, the cells were treated with DTX, CA, or nanoformulations at various concentrations (0.625, 1.25, 5, 10, 20, 40 ng/mL) for 48 h. The cells treated with a cell culture medium were used as a control. Finally, the absorbance of the cultures was detected at 450 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Scientific Inc. Waltham, MA, USA).

RAW 264.7, U2OS, 4T1, Ishikawa, MC 38, Panc 02, 143B, and SW480 cells were cultured in 96-well plates (10⁴ cells/well) and incubated overnight. To evaluate the cytotoxicity of PA and MnO₂@PA NPs, cells were incubated with different concentrations of PA and MnO₂@PA NPs (0, 12.5, 25, 50, 75, 100, 125, 150 μg/mL) for 4 or 24 h. After incubation, cells were washed with phosphate-buffered saline (PBS) to remove PA and MnO₂@PA NPs, and 100 μL medium containing 10% CCK-8 solution was added to each well with another 30-min incubation, followed by optical density was measured at 450 nm using a Thermo Multiskan Spectrum spectrophotometer (Varioskan Flash, Thermo Scientific Inc., USA).

The TBA value was determined using the distillation method [23 (link)] with slight modifications. Two grams of crushed FD tofu was suspended in 38 mL of 0.2 M HCl containing 0.2% butylated hydroxytoluene (0.2 mL) and distilled until the total volume reached 50 mL. A 3 mL of the aliquot was mixed with 3 mL of 5 mM 2-thiobabituric acid (dissolved in 50% acetic acid; Sigma-Aldrich, St. Louis, MO, USA), reacted at 90 °C for 30 min, and the optical density was measured at 538 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Scientific, Waltham, MA, USA). The TBA value was determined using a standard curve of 1.56–25 µg/mL malonaldehyde bis (MA; Sigma-Aldrich), and the value was calculated as milligrams of equivalent MA per kilogram of the sample.

Mouse breast cancer cell line 4T1 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 4T1 cells were incubated in DMEM cell culture supplemented with 10% (v/v) FBS, streptomycin (100 µg/mL), and 100 IU penicillin at 1 × 10⁴ cells/well in 96-well plates at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h. To evaluate the cytotoxicity of Lut, blank NPs, and nanoformulations, 4T1 cells were co-incubated with Lut or its nanoformulations at various concentrations (5, 10, 20, 40, 60, 80, or 100 μM) for 48 h. Otherwise, cells were incubated with equal blank NPs to investigate the cytotoxicity of blank NPs. After incubation, the culture supernatant was discarded, and 110 µL of CCK-8 culture solution was added to each well and cultured for another 0.5 h. Subsequently, the absorbance of the cultures was measured at 450 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Scientific Inc. MA, USA).

The cytotoxic activity of TL on the cultured cells was measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric test [19 (link)]. The cells were grown in 96-well plates at a density of 1 × 10⁵ cells/mL. After overnight incubation, the cells were treated with different concentrations (from 10 to 100 µM) of the test compound and incubated for 24 and 48 h. Later, the MTT solution (20 µL of 5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and the plates were re-incubated for an additional 4 h. Finally, the medium was removed and 100 µL of lysis buffer (10% SDS, 0.01 M HCl) was added to solubilize the formed formazan crystals. The amount of formazan crystal was determined by measuring the absorbance at 570 nm using a Thermo Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the half-maximal inhibitory concentration (IC₅₀) of the pure compound was calculated by GraphPad software. All experiments were performed in triplicate, at least three times independently.