Biotek synergy microplate reader

Mitochondrial complexes’ activities were assessed as previously described [29 (link)]. Briefly, CI oxidizes NADH while reducing DUB to DUbH₂, which is then oxidized by DCPIP. Therefore, the decrease in the absorbance of DCPIP at 600 nm is proportional to the activity of CI. CII activity was measured in presence of 2.5 µM rotenone, using the CII’s capacity of reducing DUB. Therefore, the decrease in the absorbance at 600 nm is proportional to the activity of CII. The activity of CIII was evaluated using the increase in absorbance at 550 nm (cytochrome c reduction) by addition of DUbH₂. The activity of CIV was evaluated by addition of 0.5 mM TMPD plus 2 mM ascorbate to respiration medium MiR05 supplemented with 0.5 µM rotenone plus 2.5 µM antimycin A. Oxygen consumption rate was proportional to CIV activity. ATP synthase activity was measured following the reduction of NADP⁺ at 340 nm using an enzyme-linked assay [18 (link)]. Absorbance measurements were performed at 37 °C using a Synergy-Biotek microplate reader (Biotek Instruments, Winooski, VT, USA) and the oxygen consumption rate measurements were performed using a high-resolution respirometer (Oxygraph O2k). The specific activity of each complex was determined by the subtraction of the activity in the presence of the appropriate inhibitor from the non-inhibited one. The results are expressed as nmol/min/mg protein.

Cell viability was determined after 24 h of incubation by MTT assay, as previously described [55 (link)]. The examined extracts were dissolved in DMSO, and then diluted in the culture medium to the appropriate concentrations (from 0 to 500 μg/mL). The absorbance was measured at 570 nm using a Biotek Synergy microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Three independent experiments were performed, and the results are expressed as cell viability as % of the control, untreated cells (mean ± SD), and IC₅₀ values (concentration at which viability is inhibited by 50 percent).

Cells were plated on 96‐well plates. After 2 days of cell growth, 10 µL/well of WST‐8 reagent (cell counting kit 8; Sigma‐Aldrich, 96992) was added and absorbance at 450 nm was measured by using a BioTek Synergy microplate reader (BioTek Instruments). Medium without cells was used as background, and the A₄₅₀ of background was subtracted from the samples.

Human cancer and the corresponding normal cell lines, used in the study, were grouped as follows: skin panel (skin keratinocytes HaCaT, malignant melanoma: A375 and WM793), prostate panel (prostate epithelial cells PNT2, prostate carcinoma: DU145 and PC3), and gastrointestinal panel (hepatocellular carcinoma HepG2, colon adenocarcinoma: Caco-2 and HT-29). Cells were grown at standard conditions (37 °C, 5% CO₂, relative humidity) and culture media (DMEM/F12 for PNT2, WM 793, HT-29, PC3; DMEM Low Glucose for DU145; DMEM High Glucose for A375, HaCaT; MEM with NEAA for Caco-2), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics solution (10 000 U penicillin and 10 mg streptomycin/mL). All culture media and supplements were from Sigma-Aldrich. Before the experiment, cells were seeded onto 96-well plates for 24 h (1.5 × 10⁴ cells/well). The culture medium was replaced with fresh medium containing different concentration of the tested extracts (50–500 μg/mL) and incubated for 24 h. Cell viability was measured with the lactate dehydrogenase (LDH) assay (Clontech, Mountain View, CA, USA), as described previously [27 (link)]. The absorbance was measured at 490 nm using a Biotek Synergy microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Cell viability was expressed as % of control (untreated cells). Each experiment was done in triplicate.