Dmi1 inverted microscope

Biofilms were assayed using the microplate method with crystal violet staining as described previously (Djordjevic et al., 2002 (link)). Briefly, 200 μl of diluted (1:100 in BHI broth) bacterial culture (bacterial concentration of approximately 10⁴ CFU/ml) were transferred into microtiter plate (Corning). The plates were statically incubated at 37°C for 24 h, 48 h and 72 h. To assess the number of planktonic cells, the cultures (100 μl) were centrifuged, and the pellets were resuspended in 1 ml of sterile saline. The bacterial cultures were then serially diluted and 100 μl volumes were taken for colony counting. To quantify the biofilm production, the medium was removed after incubation and then the wells were gently washed five times with sterile water. Biofilms were stained with 1% crystal violet for 45 min and washed with sterile water. Finally, biofilms were decolorized by 95% ethanol. The absorbance at OD_{595 nm} was measured to determine biofilm production. Finally, the biofilms were visualized under a DMi1 inverted microscope (Leica-Microsystems, Wetzlar, Germany).

Endothelial EA.hy926 cells were transferred to 24-well culture plates and cultured until confluent. The cells were treated with arsenite (NaAsO₂, Fujifilm Wako Pure Chemical Co., Ltd.) at 1, 2, 5, 10, or 20 µM and incubated at 37 °C for 24 or 48 h. After treatment, the medium was discarded, and the cells were washed twice with Dulbecco’s phosphate-buffered saline (D-PBS, Fujifilm Wako Pure Chemical Co., Ltd.). The cells were fixed with methanol and stained with Giemsa solution (Merck KGaA, Darmstadt, Germany). The cell layer was observed morphologically using a DMi1 inverted microscope (Leica Microsystem, Wetzlar, Germany). Separately, cell viability was measured using MTT (Dojindo Laboratories, Kumamoto, Japan). Briefly, after treatment with arsenite, the culture medium was changed to fresh 10% FBS-DMEM containing 0.5 mg/ml MTT, and cells were incubated for 4 h at 37 °C. After removing the medium, dimethyl sulfoxide (Fujifilm Wako Pure Chemical Co., Ltd.) was added to MTT formazan. Absorbance at 570 nm was measured by a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

The influence of NLCs on cell viability was also studied under stress conditions for BJ fibroblasts. They were seeded in a 96-well plate at a concentration of 50,000 cells/well (156,000 cells/cm²) in 10% FBS DMEM medium. Plates were incubated for 24 h at 37 °C and 5% CO₂. Medium was taken off and cells were washed with 200 µL of PBS. After 24 h incubation with 10 µM CUR-NLCs, 0.54 g/L Blank-NLCs or Trolox at 10 µM in 10% FBS DMEM, medium was removed and cells were washed with 200 µL of PBS. Then, cells were treated with Luperox^® at 100 μM for 1 h. Afterwards, Luperox^® was removed and the MTT test was performed as in no stress conditions: 200 µL of 0.5 g/L MTT in PBS was added to experimental wells and then plates were incubated for 2 h 30 min (37 °C and 5% CO₂). After incubation, photos were taken using a DMi1 inverted microscope (Leica Microsystems, Wetzlar, Germany). Obtained RGB images were converted to grayscale images using Fiji software (ImageJ, NIH, USA). MTT was then removed and 200 µL of isopropanol was added to each well. Plates were gently stirred and stored at room temperature in the dark for 40 min. The percentage of viable cells was calculated as in no stress conditions through absorbance readings at 570 nm as described in Section 2.8.1 with a microplate reader (Xenius XM, Safas, Monaco).

The cell shape was observed using bright‐field microscopy using a DMi1 inverted microscope (Leica Microsystems, Wetzlar, Germany). Cell proliferation was examined using the CellTiter 96 aqueous one‐cell proliferation assay kit (Promega, Madison, USA) as per the manufacturer's instructions. The absorbance was read with a plate reader (SpectraMax M2e, Molecular Devices, San Jose, USA) at 490 nm.

Cell migration assays were performed using a culture-insert 2 well in µ-Dish (Ibidi GMBH, Gräfelfing, Germany). According to the manufacturer’s protocol, 7 × 10⁴ cells were seeded in culture-insert dishes and incubated until they reached confluency. Then, the plastic inserts were removed and the dishes were washed with PBS and re-equipped with 1 mL McCoy’s 5A complete medium (Life Technologies, Paisley, UK) before being incubated at 37 °C to allow for wound closure. Images were taken by a LEICA DMi1 inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) at 0 h, 24 h, and 48 h.