Calcein am pi double staining kit

To evaluate the photothermal response of Au‐Ur@DTTC nanoparticles, 0.5 mL solution of these nanoparticles were transferred into a quartz cuvette and the temperature was measured every minute (until 10 min) using a digital thermometer that was connected to a thermocouple device. The top of the cuvette was sealed with a cap to reduce heat loss and evaporation from the surface of the solution.
To evaluate the in vitro cell killing caused due to nanoparticles photothermal effect, the SKOV3 cells (10000 cells per well of a 96‐well plate) were incubated with Au‐Ur@DTTC (0.15 mg mL⁻¹) for 24 h. PTT was performed under 808 nm laser (MDL‐N‐808‐10W, Changchun, China) for 5 min (spot size 5 mm, 0–1.5 W cm⁻²). The penetration depth tests were conducted by covering different thicknesses of chicken tissues (0, 2, 4, and 6 mm) on 96‐well plate. Following these treatments, cell viability was performed by MTT assay kit (YEASEN, Shanghai, China). Live/dead cells were stained using a Calcein‐AM/PI double staining kit (YEASEN, Shanghai, China), and imaged with fluorescence microscopy (20× magnification). The measurements were taken thrice 24 h post laser irradiation (n = 3).

A Calcein-AM/PI double staining kit (Cat no.: 40747ES76, Yeasen, China) was used to detect live/dead cells. Briefly, 1 × 10³ cells (SCC9) were seeded in glass bottom culture plates. After 24 h of culture, cells were cultured in a neutral environment, in a neutral environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄, in an acidic environment, and in an acidic environment supplemented with 100 μg mL⁻¹ Zn_0.4Mg_0.6Fe₂O₄ for an additional 24 h. The cells were then washed with 1× assay buffer three times and incubated with 100 μL of detection buffer (5 μL of 2 mM Calcein-AM solution and 15 μL of 1.5 mM PI solution in 5 mL of assay buffer) for 15 min at 3 °C and observed under a confocal scanning system.

HCECs were inoculated in a 24-well plate at a density of 1 × 10⁴ cells/well and cultured overnight. Then, the free drug and the FK506 liposomes were added at different concentrations (0, 5, 25, and 35 μg/ml) and incubated for 24 h. Subsequently, the drug solution was removed, and the cells were washed 3 times with PBS. After that, the cells were stained using a Calcein-AM/PI double staining kit (Yeason, China) (Calcein-AM, 0.67 μM; PI, 1.5 μM) and incubated at 37°C for 15 min in the dark. Finally, an inverted fluorescence microscope (Zeiss, Germany) was used to record the images.

After removing the mesh samples the cells were washed with 1X PBS buffer and then stained using the Calcein-AM/PI double staining kit (Yeasen) according to the manufacturer’s instructions. 200 ul staining solution (final concentration of 2 μM Calcein-AM and 4.5 μM PI) was added to each well and incubated in a cell incubator for 15 min. Afterward, the stained cells were rinsed with 1X PBS buffer and then examined with an inverted microscope. Photographs were obtained under a fluorescence microscope (model) using emission filters of 490 ± 10 and 545 nm, respectively.

TMZ and L-GSH were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Methylene blue (MB) and 2,7-dichlorofluorescein diacetate (DCFH-DA) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Dopamine hydrochloride (DA‧HCl), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), and indocyanine green (ICG) were purchased from Aladdin (Shanghai, China). 4’,6-Diamidino-2-phenylindole (DAPI), Lyso-Tracker Red, and Hoechst 33,342 were obtained from Beyotime (Shanghai, China). D-luciferin potassium salt was purchased from BioFroxx (Germany). Calcein-AM/PI double staining kit was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Annexin V-FITC/PI detection kit was purchased from Becton, Dickinson and Company (USA). The Luciferin (Luc) lentiviral vector was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd (GV260 vector, Shanghai, China). MGMT enzyme linked immunosorbent assay (Elisa) kit was obtained from Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China).

Tetrabutyl titanate were obtained from Aladdin (Shanghai, China). (3‐Aminopropyl) trimethoxysilane (APTMS) was purchased from Macklin (Shanghai, China). Titanium butoxide (GC, 99.0%), hydrogen peroxide (H₂O₂), heptanoic acid (AR, 98.0%), ethanol, N‐(3‐dimethylaminopropyl)‐N‐ethylcarbodiimide hydrochloride (EDC), doxorubicin (DOX) and N‐hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). Cantharidin (CTD) was purchased from Sigma (St. Louis, MO, USA). NHS‐PEG₂₀₀₀‐COOH was purchased from Ponsure Biotechnology Co., Ltd (Shanghai, China). The BCA Kit and 4′,6‐Diamidino‐2‐phenylindole (DAPI) were obtained from Solarbio (Beijing, China). Cell Counting Kit‐8 (CCK‐8) and DCFH‐DA were purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). Fetal bovine serum (FBS), RPMI 1640, DMEM, trypsin‐EDTA and penicillin‐streptomycin were obtained from Zhejiang Tianhang Biotechnology Co. Ltd (Zhejiang, China). The YSA peptide (YSAYPDSVPMMSK) was obtained from GL Biochem Peptide Co., Ltd (Shanghai, China). The calcein‐AM/PI double staining kit was obtained from Yeasen (Shanghai, China). Annexin V‐FITC/PI apoptosis double staining kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China). The abbreviations are summarized in Table 1.