Elx800 absorbance microplate reader

Cells (1 × 10⁵/mL) were inoculated into 96-well plates. The CCK-8 assay was performed at 24 h, 48 h, 72 h, and 96 h, according to specific protocols. Finally, the ELx800 Absorbance Microplate Reader (BioTek) was utilized to measure the colored solution at the wavelength of 450 nm.

Seminal plasma TAC measurement was operated according to the antioxidant assay kit (Cayman Chemical) that is similar to previous reports (Mahfouz, Sharma, Sharma, Sabanegh, & Agarwal, 2009; Miller & Rice‐Evans, 1997). The assay measures the ability of total antioxidants in the seminal plasma to inhibit the oxidation of the 2,20‐Azino‐di‐[3‐ethylbenzthiazoline sulphonate] (ABTS) to ABTSt. The antioxidant capacity to inhibit ABTS oxidation in the sample was compared to that of a water‐soluble tocopherol analogue, Trolox. ELx800 Absorbance Microplate Reader (BioTek Instruments, Inc.) was used to monitor absorbance at 750 nm.

The FPA cells (GT1-1, GH3) were plated into 96-well culture plate with a density of 500 cells/well, and were treated with different doses of T-5224, RSPO2, and AZD5363, respectively, as well as STO-609 (CaMKK inhibitor) and Genipin (aglycone derived from the iridoid glycoside), both of which protect against several types of tumors, including brain tumors. We used MTT (Sigma, St. Louis, Missouri, USA) dissolved in PBS (5 mg/ml) to measure the viability of cells. On the day of measurement, the medium was replaced on fresh DMEM supplemented with 10% FBS and diluted MTT (1:10, 10% MTT), and incubated for 3.5 h at 37 °C. Then, the incubation medium was removed and formazan crystals were dissolved in 200 μl solution of DMSO. We used an ELx800 absorbance microplate reader (BioTek Instruments, VT, USA) to quantify the MTT reduction by measuring light absorbance at 570 nm. Each test was repeated four times.

Scavenging activity of DPPH radical was conducted according to Brand-Williams et al., [63 (link)] with slight modifications. DPPH solution was prepared in methanol at 0.2 mM. Then, 100 µL samples were mixed with 100 µL of DPPH solution. The sample mixture was kept at room temperature in a dark place for 30 min. The absorbance was measured at 515 nm using ELx800 Absorbance Microplate Reader (Biotek Instrument, USA). Ascorbic acid (3.125–100 µg/mL) was used as a standard. The negative control for this analysis was the DPPH solution in the absence of sample. The percentage of free radical scavenging for both the DPPH and ABTS radical scavenging assay was calculated using Equation (4).

Cupric ion reducing antioxidant capacity (CUPRAC) assay was evaluated by some modifications in the method of Apak et al. (2004) [76 (link)]. Briefly, 10 μL of samples and 190 μL of CUPRAC reaction solution (containing 7.5 mM neocuproine, 10 mM Cu(II) and 1 M acetate buffer (pH 7) in a ratio 1:1:1 (v/v/v)) were mixed. Reaction mixtures were then incubated at 25 ± 1 °C for 15 min and absorbance was recorded at 450 nm using a BioTek ELX800 Absorbance Microplate Reader (BioTek Instruments). Assays were made in triplicate and antioxidant capacity determined using this assay was expressed as TAEC.

Ferric reducing antioxidant power (FRAP) [16 (link)] and cupric ion reducing antioxidant capacity (CUPRAC) [17 (link)] protocols were used to evaluate the in vitro antioxidant activity of the compounds. Briefly, 10 μL of the extracted sample was mixed with 190 μL of FRAP (10 mM TPTZ; 20 mM FeCl₃·6H₂O and 300 mM acetate buffer pH 3.6; ratio 1:1:10 (v/v/v)) were used for FRAP assay, whereas 10 μL of the extracted sample was mixed with 190 μL of CUPRAC (10 mM Cu(II); 7.5 mM neocuproine and 1 M acetate buffer pH 7; ratio 1:1:1 (v/v/v)) were used for CUPRAC assay. Incubation lasted 15 min at room temperature. Absorbance of the reaction mixture was measured at 630 nm for FRAP and 450 nm for CUPRAC with a BioTek ELX800 Absorbance Microplate Reader (BioTek Instruments, Colmar, France). Assays were made in triplicate and antioxidant capacity was expressed as Trolox C equivalent antioxidant capacity (TAEC).