Fluoroskan fl

Human neutrophils were isolated from peripheral blood and re-suspended in Hank’s Balanced Salt Solution (HBSS) comprising 138 mM NaCl, 5.4 mM KCl, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM MgSO₄, 4.2 mM NaHCO₃, 5.5 mM glucose, and 20 mM HEPES, pH 7.2, and dispensed into white 96-Well Immuno Plates (2 × 10⁵ cells/well). Neutrophils were incubated with 500 μM Luminol (Sigma) and different concentrations of Alb for 30 min at 37 °C. For the C6 group, neutrophils were pre-incubated with 20 μM C6 for 30 min before the incubation with Luminol and Alb. After incubation, neutrophils were stimulated with fMLF and the chemiluminescence was measured immediately every 1 min for 60 min using Fluoroskan FL (ThermoFisher) equipped with internal software SkanIt 2.6.

Rabbit mesenchymal stem cells (MSCs) were used to study the adhesion and toxicity of the surface modification of the titanium alloy plate to cells. Titanium alloy plates with different treatments were sterilized in 75% ethanol for 2 h, then washed three times with sterile PBS, and equilibrated in the culture medium for 2 h. MSCs were seeded on the plates with a density of 1×10⁵ cells per well. After 6, 24, and 48 h, the surface of each titanium alloy plate was washed three times with PBS to remove any loosely attached cells. Then, 0.5 mL of 10% CCK-8 medium was added to each well and they continued to incubate for 1 h, then the absorbance was measured at 450 nm (Fluoroskan FL, Thermo Fisher, Waltham, MA, USA). Three parallels were set for each sample. At the same time, fluorescence imaging was used to observe the adhesion of MSCs at different time intervals. The cells were stained with a Live/Dead Cell Double Staining Kit.

For indirect release study in vitro, MG63 cells (human osteosarcoma cells, ATCC^® CRL-1427) were seeded at 2500 cell/cm² density. After 24 h, hybrids were added to cells after dilution in cell medium (dilution factor equal to 10). After 1 day, cell media were collected and centrifuged (13,800× g, 5 min). The resulting pellets were included in OCT and frozen in liquid nitrogen. Serial sections of 6 μm width were obtained using a semi-automatic cryostat (MC500, Histo-line Laboratories, Pantigliate, Italy) and visualized with an inverted fluorescent microscope. Cells were fixed in 4% PFA, and the nuclei were stained with DAPI. Finally, cells were visualized with an inverted fluorescent microscope. For fluorescent lipid quantification assays, liposomes and hybrids were loaded with 56.8 and 11.4 µg/mL of fluorescent lipid, respectively. Cells were seeded in a 96-well plate at 1500 cells/well density. After 24 h, formulations were 10-fold diluted in cell media and incubated with cells. At fixed time points (3 h, 24 h, 48 h, 72 h, 7 days), fluorescent lipid uptake was evaluated by spectrofluorimetric analyses (Fluoroskan FL, Thermo Scientific, USA).

MMP was measured by rhodamine 123 (Rh123). Briefly, 1 mg isolated mitochondria were incubated with 2 ml buffer (pH 7.4) containing rotenone (2.5 μM), sucrose (125 mM), KCl (65 mM), HEPES-KOH (10 mM), and Rh123 (0.2 μM), at 30°C for 10 min. After these, the fluorescence in different groups was measured by a fluorescence microplate (Fluoroskan FL, Thermo). The results were showed as the fold of control group.

The assay was performed using the maximum non-toxic concentration (MNTC) of each natural product in HEK293-ACE2 cells. A total of 1 × 10⁴ cells in suspension were added to each well of 96-well plates. The mix (50% natural product dilution and 50% virus dilution) was added to the cells. Firefly luciferase activity was measured 48 h later using Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA) and Fluoroskan FL (Thermo Scientific). HEK293T-ACE2 cells transduced with the pseudotyped virus without any natural products were used as control untreated cells. The following formula was used to calculate the percentage of inhibition: 100 − (RLUs of treated cells/RLUs of control untreated cells) × 100. In all assays, RLUs of untreated pseudotyped-virus-transduced HEK293T-ACE2 cells were at least 10-fold over the RLUs of untransduced cells. The selectivity index (SI) was determined as the ratio of CC₅₀ versus IC₅₀.

SIRT1 activity was determined using the SIRT1 Fluorometric Assay Kit (AS-72155, AnaSpec, Fremont, CA, USA) according to the manufacturer’s instructions. Fluorescence was measured using a fluorometric reader (Fluoroskan FL, ThermoFisher Scientific, Waltham, MA, USA) with excitation at 490 nm and emission at 520 nm.

Specific volumes (100 µL) of different concentrations of ZnO/PVP solution (1.56, 3.125, 6.25, 12.5, 25, and 50 µg/mL) were pipetted into the wells of a 96-well plate. The fluorescence intensities were measured using a fluorescence microplate reader (Fluoroskan FL; Thermo Scientific, Waltham, MA, USA) (excitation wavelength: 365 nm, emission wavelength: 570 nm) to obtain the fluorescence intensities at each concentration of the ZnO QDs.