Stat fax 3200 microplate reader

The samples were placed into 24-well culture plates and cells were seeded with a density of 8 × 10⁴ cells per ml in α-MEM supplemented with 10% fetal bovine serum (PAA Laboratories). The next day, the culture medium was replaced with α-MEM supplemented with 10% fetal bovine serum, 50 μg ml^–1 ascorbic acid, and 10 mM β-glycerophosphate (Sigma), and incubated at 37 °C in a humidified incubator containing 5% of CO₂. The medium was changed every three days. After culturing for 14 days, the samples were washed with Earle's Balanced Salt Solution and transferred into another plate. The cells were detached from samples with a trypsin/EDTA solution, washed with PBS three times, then re-suspended in 200 μl of 0.1% Triton X-100, 10 mM Tris-Cl (pH = 7.5), 1 mM MgCl₂ and underwent 4 cycles of freeze–thaw and sonication. After that, 100 μl of p-nitrophenyl phosphate (pNPP) was added to 10 μl of cell lysate followed by incubation for 30 min at 37 °C. The reaction was stopped by adding 0.4 M of NaOH and the plate was read at a wavelength of 405 nm by Stat Fax 3200 microplate reader (Awareness Technology Inc). The ALP activity was determined as a rate of p-nitrophenol liberation from p-nitrophenyl phosphate and expressed as nmol of p-nitrophenol formation per minute per milligram of cellular protein. All measurements were reported as the mean average value ± standard deviation.

The NO scavenging activity of A. viridis extracts was determined according to the method of Tsai et al. [16 (link)]. A. viridis extracts (60 μL) at various concentrations (50, 100, 150, 200, 250, and 300 μg/mL) were mixed with sodium nitroprusside (10 mM in phosphate buffered saline, 60 μL) in a flat-bottomed microtiter plate and incubated in the light for 150 min at room temperature. An equal volume of the Griess reagent was added to the wells to measure the nitrite content. A₅₄₆ was measured with a microtiter plate reader (Stat Fax 3200 Microplate Reader, Awareness Technology Inc., USA). Ascorbic acid and α-tocopherol were used as the standard antioxidants. The NO scavenging activity was expressed as % inhibition as in formula (2).