96 well plate

Extracts (11.7 µL) obtained using hexane-based extraction were loaded in a 96-well plate (Eppendorf, Hamburg, Germany) together with 15 µL 1.33 mM ammonium formate in 2-propanol. Extracts (15 µL) obtained using chloroform-based extraction were loaded in a 96-well plate (Eppendorf, Hamburg, Germany) and added 15 µL 0.01% methylamine. These extracts were analyzed by high-resolution Fourier transform mass spectrometry (FTMS) in negative ion mode using either an LTQ Orbitrap XL or an Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA), both equipped with a Triversa NanoMate robotic nanoflow ion source (Advion Biosciences, Ithaca, NY, USA). On the LTQ Orbitrap XL, each sample was analyzed for 5 min, using an FTMS scan range of m/z 150–420, a max injection time of 250 ms, automated gain control for an ion target of 1e5, two microscans, and target resolution setting of 100,000. On the Orbitrap Fusion Tribrid, each sample was analyzed for 5 min, using a FTMS scan range of m/z 150–420, a max injection time of 100 ms, automated gain control for an ion target of 1e5, three microscans, and target resolution setting of 500,000. The sample injection settings of the NanoMate for samples injected with ammonium formate were −0.96 kV and 1.25 psi. The NanoMate settings for samples injected with methylamine were −0.96 kV and 0.6 psi.

hUC-MSCs were seeded at 4 × 10³ cell density into a 96-well plate (Eppendorf, Hamburg, Germany) in 150 μL of DMEM/F12 medium supplemented with 10 % FBS and 1× Pen/Strep and incubated at 37 °C up to complete adhesion (about 6 h). Then, the culture medium was changed to 150 μL of each chitosan extract supplemented with 2% FBS and 1× Pen/Strep. After 24 h of incubation, the Cytotoxicity Detection Kit (Roche, Basel, Switzerland) was used to evaluate the cytotoxicity of liquid extracts of chitosan on hUC-MSCs. According to the manufacturer’s instruction, plate was centrifuged at 300× g for 5 min. Then, the 100 μL of supernatant was transferred to a new 96-well plate (Eppendorf) and the same volume of the LDH-substrate mixture was added to each well. After 30 min of incubation in the dark at the room temperature, the absorbance at 450 and 620 nm was measured by the Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA). hUC-MSCs cultured in DMEM/F12 medium supplemented with 2% FBS medium and 1× Pen/Strep was used as a control. Three independent experiments were performed. Statistical analysis was performed by one-way ANOVA and the Bonferroni test (post-hoc test) using the GraphPad Prism v.5 software.(GraphPad Software, Inc., CA, USA) p values less than 0.05 (p < 0.05) were considered statistically significant and labelled by an asterisk (*).

The cell viability was measured using the CCK-8 assay according to the manufacturer's instructions and the hydrogen peroxide induced RAW264.7 cells oxidative stress model was established following the method described by Zhou et al. with some modifications (34 (link)). RAW264.7 cells were seeded in 96-well plates (Eppendorf, Germany) at a density of 6 × 10⁴/ml in culture medium for 4 h. Subsequently, the cells were incubated with DMEM containing different concentrations of L. aureum extracts (dissolved in DMSO) or H₂O₂ for 24 h. The viability of cells stimulated with L. aureum under oxidative stress were determined as follows: cells were plated in 96-well plates (Eppendorf, Germany) with a density of 1 × 10⁵ in culture medium for 4 h, and the cells were incubated with DMEM containing different concentrations of L. aureum extracts (0, 2, 5, 10 μg/ml) for 20 h. The positive control group and the L. aureum–treated groups were then exposed to H₂O₂ (400 μM) for 4 h. CCK-8 solution (10 μl) was added to each well and incubated in an atmosphere of 5% CO₂ at 37°C for 4 h. The absorption values were measured at 450 nm by using a spectrophotometer (Epoch Microplate Spectrophotometer; BioTek Instruments). The results were expressed as the percentage viability according to the following formula: