Hoechst 33342 staining solution

SH-SY5Y cells were seeded in six-well plates with approximately 1 × 10⁵ cells per well. After being completely attached, SH-SY5Y cells were divided into control group and experimental groups, which were pretreated by different concentrations of OPA (0 μg/ml, 50 μg/ml, 100 μg/ml, and 500 μg/ml) for 24 h prior to treatment with 0.5 mM MPP⁺ for the other 24 h. Next, SH-SY5Y cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min and then washed three times with PBS. After incubation by a Hoechst 33342 staining solution (Beyotime, China) for 3–5 min at room temperature, PBS-rinsed SH-SY5Y cells were observed under the fluorescence microscope. Compared to normal cells, the apoptotic cells exhibited brighter fluorescence and densely stained nuclei.

The selenium powder (99.5%, 325 mesh, Alfa Aesar), sodium ascorbate (99%, Adamas-Beta), PVP (MW = 40,000, K30, Sigma), SnCl₂·2H₂O (99.99%), NaBH₄ (98%), and HAuCl₄·3H₂O (99.9%) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Methanol (GR) and K₂CO₃ (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. Luciferase isothiocyanate (FITC, ≥90%), 3, 3′, 5, 5′-tetramethylbenzidine (TMB), and methylene blue (MB, 95%) were purchased from Sigma-Aldrich Co., Ltd. DCFH-DA and Hoechst 33342 staining solution were purchased from Beyotime Co., Ltd. All chemical agents in this work were utilized without further purification.

Urea, arginine, and glycerol were purchased from MACKLIN; Tris and ethylenediaminetetraacetic acid (EDTA) were purchased from Biofroxx; sodium chloride was purchased from Guangdong Guanghua Sci-Tech Co., Ltd.; dithiothreose alcohol, 3, 3', 5, 5' - tetramethylbenzidine (TMB) substrate for ELISA, ELISA stopping solution, glue-activated horseradish peroxidase labeling kit, and Triton X-100 were purchased from Solarbio; Freund’s complete adjuvant, Freund’s incomplete adjuvant, HAT, and HT were purchased from Sigma; goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) was purchased from Sango Biotech; fetal bovine serum and RPMI 1640 medium were purchased from ThermoFisher Scientific; PBS was purchased from GBCBIO Technologies; saturated ammonium sulfate solution was purchased from Leagene; PAGE gel quick preparation kit was purchased from ATGene; and non-reducing loading buffer and reducing loading buffer were purchased from GBCBIO Technologies, Inc. Confocal dishes were purchased from NEST Biotechnology Co., and 4% paraformaldehyde, goat serum, Hoechst 33342 staining solution, and anti-fluorescence quenching sealer were purchased from Beyotime. Fluorescein (FITC)-conjugated goat anti-mouse IgG (H + L) was purchased from Proteintech.

The slides of treated cells were fixed with 4% paraformaldehyde for 30 min, and then Hoechst 33342 staining solution (Beyotime, China) was added away from the light for 5 min. After being washed and sealed, the cells were captured with a fluorescence microscope.
To assess cell apoptosis by flow cytometry, treated cells were collected, resuspended in binding buffer and then stained with PE Annexin V and 7‐AAD (BD Biosciences). The cells were analysed by an Accuri flow cytometer (BD Biosciences). Flow‐Jo software was used to analyse the results.

Cell proliferation was detected with the BeyoClick™ EdU-555 Cell Proliferation Assay Kit (C0075S, Beyotime). The cells were incubated in 6-well plates, and EdU working solution (20 μM) pre-warmed at 37 °C was added to the 6-well plates for a 1-h incubation. After the EdU labeling of cells was completed, the culture medium was removed. The cells were fixed by adding 1 mL of 4% paraformaldehyde for 15 min, incubated with phosphate-buffered saline (PBS) permeabilization solution for 10–15 min, and with 0.5 mL of Click reaction solution for 30 min in the dark (all at room temperature). Cell nuclei were stained with Hoechst 33,342 staining solution (C1025, Beyotime), and then cell proliferation was observed under a fluorescence microscope.
CD8⁺ T cell proliferation in the co-culture system was assayed with the CellTrace™ CFSE Cell Proliferation Kit (C34554, Thermo Fisher), and CFSE density was measured by flow cytometry.