Az100 fluorescence microscope

The generation of intracellular ROS was determined using MAK 142 fluorometric kit (Sigma Aldrich, USA). A549 and MDA cells were seeded at a density of 5 × 10⁴ cells in black 96-well plates with clear bottom. In a preliminary study, the ROS generation peaked at 120 min, thus taken as the incubation time. Cells were treated with IC₅₀ and 10-fold IC₅₀ doses of respective treatments (Cur, GP-Cur, Ptx, GP-Ptx, GP-Cur-Ptx) and GP (0.15 mg/ml) as well as plain media (untreated) cells served as negative controls. Following incubation for 120 min, the ROS generated were determined according to the manufacturer’s protocol. Subsequently, the fluorescence intensity was quantified using a fluorescence Varioskan flash microplate reader (Thermo scientific, USA) with excitation and emission wavelengths fixed at 650 and 765 nm, respectively. ROS generation was determined as the percentage of ROS compared to control. For fluorescence microscopy imaging, A549 and MDA cells were seeded in 4-well chamber slides (SPL Life Sciences, South Korea). ROS reagents of 125 μL were used and images were captured using Nikon Az100 fluorescence microscope (Nikon, Japan).

A549 and MDA cells were seeded at a density of 5 × 10⁴ cells in 4-well chamber slides and treated with IC₅₀ and 10-fold IC₅₀ doses in comparison with an untreated control and GP (0.15 mg/ml) for 24 h. Thereafter, apoptosis was detected by using APOAC Annexin V-Cy3 apoptosis detection kit according to the manufacturer’s protocol (Sigma Aldrich, USA) with slight modifications. Briefly, treated cells were washed twice with PBS (100 μL) and three times with 100 μL of binding buffer. Subsequently, 100 μl of double-label staining solution containing Annexin Cy-3 and 6-Carboxyfluorescein diacetate (6-CFDA) was then added and incubated for 10 min at RT to quantify the living cells from apoptotic and necrotic cells. The staining solution was then removed and the cells were washed five times with 50 μL of binding buffer. Finally, 50 μL of binding buffer was placed in each well and covered with cover slips and images were captured using Nikon Az100 fluorescence microscope fitted with TRITC filter (Nikon, Japan). Annexin Cy-3 stains red on necrotic cells, by binding to phosphatidylserine, which is present outside the plasma membrane of cells undergoing apoptosis. Upon entering living cells, the non-fluorescent 6-CFDA is hydrolyzed by esters producing a green-fluorescent product. Cells in the early stages of apoptosis, however, stained yellowish-green53 (link).

Alizarin red S (AR-S), a fluorescent dye, could bind with a mineralized nodule in the bone matrix. AR-S labelling could reflect the degree of bone mineralization. Zebrafish larvae aged 8 dpf were dyed with 0.2% AR-S solution for 2 h. Then, they were washed with E3 water for 15 min on a shaking table at 50 rpm (repeated three times). Fluorescence images of the AR-S-stained vertebrate column were captured under an AZ100 fluorescence microscope (Nikon, Tokyo, Japan). The integral optical density of vertebrae was measured by ImageJ analysis software (Bethesda, MD).