Dmi3000b

The HeLa cells were incubated with different concentrations of the indolizinyl-pyridinium salts–β-cyclodextrin inclusion complexes (at concentrations equal to 52 μM, 70 μM, 87 μM) and imaged 15 min and at 24 h with an inverted microscope Leica DMI 3000 B equipped with a fluorescence GFP filter. Subsequently, the cells stained with the indolizinyl-pyridinium salt–β-cyclodextrin inclusion complex were treated with 65 nM solution of LysoTracker Red DND-99 (Invitrogen) for 30 min at 37 °C in 5% CO₂. The cells were then washed three times in PBS and imaged in fresh cell culture medium using an inverted microscope Leica DMI 3000 B equipped with GFP and N2.1 filters.

The HeLa cells were incubated with different concentrations of the indolizinyl-pyridinium salts and β-cyclodextrin inclusion complexes (at concentrations equal to 52 μM, 70 μM, and 87 μM) for 24 h and then imaged at 15 min and at 24 h with an inverted microscope Leica DMI 3000 B equipped with a fluorescence GFP filter. Subsequently, the cells stained with the indolizinyl-pyridinium salt and β-cyclodextrin inclusion complex were treated with 100 nM solution of MitoTracker^® Red CMXRos for 30 min at 37 °C in 5% CO₂. After staining, the cells were washed three times in PBS and imaged in fresh cell culture medium using an inverted microscope Leica DMI 3000 B equipped with GFP and N2.1 filters.

Cell adhesion and morphology were imaged using a scanning electron microscope (JEOL JSM-6390LV, Jeol, Akishima, Tokio, Japan), after crosslinking in 2.5% GA, dehydration with ethanol and ethanol/hexadimetylosiloxan solutions. For fluorescent imaging of actin cytoskeleton and nucleus, samples were fixed with 3% PFA solution for 30 min and then stained with Alexa Fluor 488 dye (ActinGreen™, Invitrogen^TM) and Hoechst 33342 (NucBlue™ Reagent, Invitrogen^TM). The observations were carried out using a fluorescent microscope (Leica DMI3000B, Leica Microsystems, Wetzlar, Germany). The live/dead cell imaging was done using LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen^TM). After 1 and 3 days of cell culture, the samples were rinsed with PBS and incubated for 15 min in calcein and ethidium homodimer-1 solution. Finally, samples were again rinsed with PBS and imaged using a fluorescent microscope (Leica DMI3000B, Leica Microsystems). The calcein interacts with live cells (stained green), and ethidium homodimer-1 interacts with dead cells (stained red).

Intracellular ROS generation was determined by H₂DCFDA assay. Briefly, cultured cells were fixed with 4% paraformaldehyde for 15min at room temperature. Fixed cells were incubated for 150 min at 37 °C with a 10 μM H₂DCFDA solution in PBS labelled with fluorescein intracellular H₂O₂. Intracellular ROS generation was observed by fluorescent microscopy (Leica DMI 3000B, Wetzlar, Germany). To quantitatively measure ROS levels, relative fluorescence was determined by a fluorescence microplate reader at excitation and emission wavelengths of 485 and 535 nm, respectively.

The dissolution products of the membranes were prepared for the migration assay. One gram of each kind of membrane was soaked in 10 mL DMEM and incubated for 24 h, and the resultant solution was obtained. HDFs were seeded in 24-well plates at 4 × 10⁵ cells per well and cultured in an incubator humidified at 37 °C with 5% CO₂. After 24 h of culture, a scratch was made with a 200-μL pipette tip at the bottom of each well followed by washing the cells with PBS. The culture medium was then replaced with PLGA, PLGA-CS and PLGA-HACC dissolution products in order to determine the effect of the materials on HDF migration. The cells cultured with DMEM (without FBS) were regarded as a control. After being cultured for 12 h, the cells were fixed with 4% paraformaldehyde for 30 min, and after being washed twice with PBS, the cells were observed and images taken with an inverted microscope (Leica DMI 3000B, Wetzlar, Germany). In addition, a statistical analysis of the HDF migration assay was performed. We measured the original width and final width of the scratches in the two groups, and the percentage of scratch shrinkage was calculated using a previously reported method [43 (link)]:

The aim of the immunofluorescence assay was to detect Nrf2 protein localization in bMECs. Briefly, cells were seeded at a 1 × 10⁵ cell density per well for 24 h in 6-well plates and then treated as mentioned earlier in the Treatment methods for cells section. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After washing three times with PBS for 10 min each, the cells were incubated with the primary antibody (anti-Nrf2; Proteintech, China) at 4 °C overnight. Then the cells were washed three times with PBS and were incubated in the secondary antibody (goat anti-rabbit IgG [H + L] Alexa Fluor-488, 33106ES60, Yeasen, Shanghai, China) for 2 h at room temperature protected from light. After washing three times with PBS, the cells were incubated with DAPI solution for nuclear staining at room temperature for 15 min in the dark. Finally, the fluorescence of the cells was observed under an inverted fluorescence microscope (Leica DMI 3000B; Germany).