Eclipse ts2r

As described in our previous study [31 (link)], 5 × 10⁴ hMSCs were cultured in NH OsteoDiff® Medium (Miltenyi Biotec, Germany) for osteogenic differentiation. The cells were cultured for 28 days with media replacement every three days. At the end of the culture, the cells were fixed with 4% paraformaldehyde, stained with a 40 mM alizarin red S solution (Sigma-Aldrich, USA) for 20 minutes at room temperature, and viewed under an inverted microscope (Nikon ECLIPSE Ts2R, Japan). For adipogenic differentiation, 5 × 10⁴ hMSCs were cultured in NH AdipoDiff® medium (Miltenyi Biotec, Germany) for 28 days with media replacement every three days. At the end of the culture, the cells were fixed with vaporized 37% formalin for 10 minutes at room temperature, stained with 0.5% (w/v) oil red O (Sigma-Aldrich, USA) in 6% (v/v) isopropanol for 20 minutes at room temperature and observed by light microscopy.

As this study is intended to evaluate the therapeutic potential of C. albicans for its application in oral candidiasis, safety evaluation was further evaluated with the HBEC. HBECs were collected from the healthy individuals with suitable oral hygiene by gently rubbing a sterile swab on the mucosal surface of the cheeks. Subsequently, the sterile swabs were suspended in sterile PBS and used immediately. The suspended HBECs were further centrifuged, and the pellets were washed thrice with sterile PBS (Souza et al., 2018 (link)). A suspension with 5.0 × 10⁵ cells ml^–1 was prepared by counting the cell suspension through Automatic cell counter (Countess II FL, Invitrogen, United States). HBECs were then incubated with different concentrations of piperine (8, 16, 32, 64, and 128 μg ml^–1) and incubated for 20 min at 37°C. Hydrogen peroxide was used as positive control. After 20 min of incubation, the cells were stained with crystal violet and visualized under microscope (Nikon Eclipse Ts2R, Japan) to assess morphological variations due to effect of the compound.

The capacity of the astrocyte migration was detected by scratching confluent astrocyte monolayers, as described previously 28 (link). A scratch was conducted in confluent primary astrocytes using a sterile 10-μl pipette tip across the surface. The cells were washed twice and maintained for an additional 24 h in a culture medium supplemented with the following treatment: vehicle PBS and EVs (1 µg/mL). The movement images of primary astrocytes were taken under light microscopy (Eclipse Ts2R; Nikon, Japan). By determining the mean migration distance of the leading cells in the scratched area; the results were quantified.

The cells were transferred to slides (CultureWell Chambered Coverglass for cell culture, Invitrogen, C37000). Cells were synchronized in serum free media and treated with rapamycin and bafilomycin A1. After that they were fixed in 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) three times. The permeabilizing of cells were performed with 0,25% Triton-X (in PBS) for 10 min and then cells were washed three times in PBS for 5 min. The cells were blocked and incubated with antip62 (Cell Signaling, 7695S) according to the antibody protocol. Cells were washed with PBS and incubated with Alexa Fluor 488 conjugated anti-rabbit (Cell Signaling, 4412S) for 1 h. After washing the nuclei were staining with DAPI (1:10,000) for 5 min and then the cells were washed again. The slides were mounted with FluorSave Reagent (Millipore, 345,789) and observed under a fluorescence microscope (Nikon Eclipse Ts2R)^{23 (link)}. At each experimental step 4–6 different images were randomly photographed with similar settings. Total number of cells was counted by nuclei staining. The well demarcated, sharp p62 dots were counted in 80–100 cells using the program QuPath-0.2.1, which is freely available from https://github.com/qupath/qupath/releases/tag/v0.2.1.

Cells were then stained with filtered Oil Red O working solution and followed the method reported previously. (Konieczny and Emerson Jr 1984 (link)). Staining images were photographically produced using a Nikon DS-Fi3 digital camera mounted on a Nikon Eclipse TS 2R light microscope. 100% isopropanol was used to reduce background signal (Cheung et al. 2015 (link)).

To evaluate the combined PTT and chemotherapy of BSA-IrO₂@DOX NPs, Saos-2 cells were seeded in 96-well plates (1 × 10⁴ cells/well) and incubated for 24 h. The cells were incubated with different concentrations of DOX, BSA-IrO₂ and BSA-IrO₂@DOX. After that, part of cells treated with BSA-IrO₂ and BSA-IrO₂@DOX were irradiated with an 808 nm laser (1.0 W cm⁻², 5 min) after 6 h incubation. The cells were then incubated for totally 24 h and the cell viability was measured by using the CCK-8 proliferation assay.
The in vitro antitumor efficiency of BSA-IrO₂@DOX NPs was evaluated by cell live/dead assays. Saos-2 cells were seeded onto 6-well plates (5 × 10⁵ cells per well) and treated as mentioned previously. After that, the cells after different treatments were washed with PBS, stained by calcein-AM (5 μg mL⁻¹) and PI (10 μg mL⁻¹) and incubated for 30 min at 37 °C. Then, the cells were washed with PBS for three times and imaged by an inverted fluorescence microscope (Nikon ECLIPSE Ts2R).

Based on our previous report (41 (link)), formalin-fixed paraffin-embedded tissues were cut, deparaffinized, and rehydrated. The tissue sections were heated in 10 mM sodium acetate (pH 9.0) for 5 min at 121°C for antigen retrieval. To remove endogenous peroxidase, tissues were bathed in a 1% (v/v) H₂O₂-PBS solution for 15 min at room temperature in the dark. The samples were then washed with 0.1% TBS-T, and blocked with 3% (w/v) FBS in PBS for 1 h. The blocked samples were incubated overnight at 4°C with the primary antibodies (diluted at 1:200). After washing three times with 0.1% TBS-T, samples were incubated with the horseradish peroxidase-conjugated secondary antibody (diluted at 1:200) for 2 h at room temperature and then washed three times with 0.1% TBS-T. The bound antibodies were identified using freshly prepared substrate buffer (0.05% [w/v] diaminobenzidine (DAB; Sigma-Aldrich) and 0.01% [v/v] H2O2 in PBS) for 5 min. After a final wash in distilled water, the sections were counterstained with hematoxylin (Bioworld, Dublin, Ohio, USA) solution for 1 min and dehydrated. Sections were examined at various magnifications using an Eclipse Ts2R (Nikon Instruments Inc., Melville, NY, USA). The ratio of the target protein-expressing cells was measured using HistoQuest software (TissueGnostics, Vienna, Austria).