Powerwave xs2

OCCM‐30 was seeded in 24‐well plates, cultured to reach 80% confluence, pre‐treated with 10 ng/mL TNF‐α for 30 minutes and mineralization induced for 7 days. The cells were lysed in 1% Triton X‐100 on ice for 30 minutes. The total protein concentration was dertermined using a BCA protein assay kit (Beyotime). Then, 20 µL of lysate of each well was incubated in a 96‐well plate with 100 µL of fresh solution of p‐nitrophenyl phosphate substrate at 37°C for 30 minutes. The reaction was quenched by the addition of 80 µL of 0.5 mol/L NaOH solution. The absorbance at 405 nm was measured using a microplate‐reading spectrophotometer (PowerWave XS2, BioTek). A standard curve describing the relationship between OD value and p‐NP concentration was generated with the additional examination of gradient diluted p‐NP. The produced p‐NP concentration in each sample was calculated using the standard curve, and the relative ALP activity was expressed as the percentage in produced p‐NP concentration per unit time per mg protein.
For ALP staining, the cells were seeded in six‐well plates, treated and mineralization induced as those for ALP activity analysis. ALP staining was performed using a BCIP/NBT alkaline phosphatase colour development kit (Beyotime Institute of Biotechnology). Pictures were taken using a light microscope and a camera.

FRAP assay was modified from that reported by Tierney et al. [5 (link)]. The oxidant in the FRAP assay consisted of a reagent mixture that was prepared by mixing acetate buffer (pH 3.6), ferric chloride solution (20 mM), and TPTZ solution (10 mM TPTZ in 40 mM HCl) in the ratio of 10:1:1. A trolox stock solution (2 mM) was prepared and diluted with ethanol to give concentrations ranging from 0.1 to 0.5 mM. Prior to analysis, the FRAP reagent was heated and protected from light, until it had reached a temperature of 37 °C. 180 μL of freshly prepared FRAP reagent at 37 °C was pipetted into a 96-well plate with either a 20 μL test sample or standard (or ethanol for the blank). The absorbance was measured at 593 nm after 50 min on an automated Power Wave XS2 microplate spectrophotometer (Bio Tek, Winooski, VT, USA). The trolox standard curve was used to calculate the antioxidant activity of the samples which was expressed as milligram trolox equivalents (TE) per milligram of sample (mg TE·mg⁻¹).

The antiradical activity (DPPH assay) of the samples was determined spectrophotometrically in a UV-Vis plate reader by monitoring the disappearance of DPPH at 517 nm, following the procedure described by Li and coworkers [51 (link)]. The radical scavenging activity was expressed as Trolox equivalents. The ORAC assay was conducted based on a previously reported method [21 (link)]. The results were expressed as μmol Trolox equivalents by using the standard curve calculated for each assay. The FRAP assay was conducted according to a previously reported procedure [21 (link),22 (link)]. Absorbance readings were taken using a visible-UV microplate reader (Power Wave XS2, Bio-Tek Instruments Inc., Winooski, VT, USA) at 593 nm. The antioxidant activities were expressed as μmol ascorbic acid equivalents.

Yeast cells in biological triplicates were back-diluted from overnight culture and were treated with various damaging agents at mid-log-phase (OD₆₀₀ of 0.5–0.7) for 30 min^{88 (link)}. The cells were then washed twice and resuspended in YPD media to a final density of OD 0.8. In all, 5 μL of cell suspensions were added to 195 μl of YPD in 96-well non-treated polystyrene microplates, in technical repeats. The plates were incubated at 30 °C with shaking, and cell density was monitored every 10 min over 24–48 h at 600 nm on a microplate scanning spectrophotometer (Biotek PowerWave XS2).
Spot assays were performed using cells cultured to mid-log phase, treated with damaging agents for 30 min at 30 °C, washed twice and resuspended to OD ~ 0.8. Threefold dilutions were then spotted on YPD plates and incubated for 2–3 days at 30 °C.