Synergy ht multi mode microplate reader

The OS (5 × 10³ cells/well) cell lines and the normal lung MRC-5 (1 × 10⁴ cells/well) cell line were plated in 96-well plates. After adhesion, the cells were treated with flavonoid compounds—10, 20, 40, 80, and 160 μmol/L—for 48 h. The WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay was performed using the Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, St. Louis, MO, USA). Briefly, after incubation with flavonoid compounds, the medium was replaced with complete medium containing 1:10 (v/v) CCK-8 reagent and the cells were incubated for 2 h at 37 °C. After incubation, the absorbance was measured at 450 nm in a Synergy HT Multi-mode Microplate Reader (BioTek Instruments, Winooski, VT, USA). For the SRB assay, after exposure, the cells were fixed for 1 h with 10% (w/v) TCA at 4 °C. Subsequently, after microplate washing with water and drying, the cells were incubated for 30 min with SRB solution (0.05% w/v in 1% acetic acid). Then, the SRB reagent was removed and the stained cells were washed four times with 1% acetic acid (v/v). After drying, 10 mM tris-base (unbuffered, pH~10.5) was added, followed by shaking for 10 min. The absorbance at 510 nm was measured in a Synergy HT Multi-mode Microplate Reader (BioTek Instruments).

For the analysis of serum corticosterone, a commercial corticosterone rat/mouse ELISA kit was used (DEV9922, demeditec diagnostics, Kiel, Germany) according to the manufacturer’s manual. The final absorbance of the colorimetric reaction was determined by a microplate reader (Synergy HT Multi-Mode Microplate Reader; BioTek, Winooski, USA) at 450nm, and concentrations were calculated after blank correction using the simultaneously assessed concentration curve from standard samples.
To analyse IL-6 and TNF-α in the plasma, commercial quantikine ELISA kits were used (R6000B for IL-6, RTA00 for TNF-α, R&D, Minneapolis, USA), again according to the manufacturer’s manual. 100μl Standard, control and samples were incubated in duplicates for 2h with 50μl of assay diluent. Samples required a 2-fold dilution because of the low amount of sample (50μl sample + 50μl Calibrator Diluent). After adding 100μl of stop solution the optical density was determined by using a microplate reader (Synergy HT Multi-Mode Microplate Reader; BioTek, Winooski, USA) set to 450nm with wavelength correction set to 540nm, and the concentrations were calculated after blank correction.

The effect of safflower extract on cell viability was evaluated using the methyl thiazolyl tetrazolium (MTT) assay [24 (link)]. Briefly, HuDe cells were seeded at a density of 5 × 10³ cells/well into a 96-well plate and incubated at 37 °C in an atmosphere of 5% CO₂. After plating overnight, 100 µL of CT aqueous extract containing increasing concentrations of polyphenols (0–150 µg GAE/mL), HSYA, or SYA (0–150 µg/mL) were added to the cell media. Cells were then incubated at 37 °C for 48 h. Then, 100 μL of MTT solution (5 mg/mL) was added to each well. The absorbance was measured at 540 nm with a Multi-Mode Microplate Reader Synergy^TM HT (BioTek Instruments, Inc.).
Moreover, we evaluated the effect of safflower extract, HSYA, or SYA on Lactate Dehydrogenase (LDH) release. LDH release, a good indicator of cellular damage, was measured with a commercially available LDH assay kit as previously described [25 (link)]. Absorbance was read at 340 nm in a Multi-Mode Microplate Reader Synergy^TM HT (BioTek Instruments, Inc.).

TPC of the S. officinalis extracts was measured using the Folin–Ciocalteu method, according to the procedure described by Kontogianni et al. with some modifications [40 (link)]. All the extracts (1 mg/mL) were dissolved in their solvent of extraction (water, ethanol, or their 50:50 mixture). Then, 200 µL of the extract was mixed with 4.8 mL of distilled water and 500 µL of Folin–Ciocalteu reagent. After 3 min, 1 mL saturated solution of Na₂CO₃ (332 g/L) was added and diluted with distilled water to 10 mL. Blank samples were prepared using 200 µL of the respective solvent without S. officinalis extracts. After 1 h, a volume of 200 µL was pipetted to a 96-well plate, and the absorbance was read at 725 nm in a microplate reader (Synergy^TM HT Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). Gallic acid (GA), in concentrations ranging from 25 to 500 mg/mL, was used to prepare the calibration curve to interpolate the content of phenols of each extract. The results are expressed as mg of GA per g of dry S. officinalis extract. All measurements were performed in triplicate for each batch of each extract.

Stock solutions of S. officinalis extracts were prepared at concentrations of 500 ug/mL in the respective buffer of each assay. AE, HE, and EE were dissolved in 25% of their solvent of extraction. S. officinalis extracts stock solutions were then serially diluted to obtain final concentrations of 5, 10, 25, 75, 125, and 250 μg/mL. For all assays, control samples without the extracts but with an equal volume of buffer were also prepared. A control with 25% of the extraction solvent was also performed to demonstrate its noninterference in the antioxidant activity assessment. The assays were performed in triplicate for each batch of each extract. A microplate reader (Synergy^TM HT Multi-Mode Microplate Reader, BioTek, Vermont, USA) was used to read either the absorbance or the fluorescence. In all antioxidant assays, the extract concentration required to inhibit 50% of the radical (half-maximal inhibitory concentration (IC₅₀)), in μg/mL, was calculated by linear or nonlinear regression of the plots presenting the extract concentration (μg/mL, in abscissa) vs. the average (%, in ordinate) of the respective radical in the solution. Lower IC₅₀ values mean the higher ability of S. officinalis extracts to neutralize the studied radicals.

Cell metabolic activity, proliferation, and recovery of WJ-MSCs after storage in 3D constructs were assessed using the resazurin assay [27 (link)]. To evaluate the cell growth within the scaffold, the metabolic activity was measured repeatedly on days 1, 4, and 7 in three identical randomly selected samples and was measured on day 8 completely in all samples. The post-storage recovery was measured after 2 or 6 days of hypothermic storage at 4 °C and 25 °C. Each sample was labelled to allow pairing of pre- and post-storage measurements. Briefly, the samples were transferred to a new 24-well plate containing 0.5 mL of fresh CCM with 40 µM resazurin in each well. The samples were incubated for 2 h at 37 °C in a humidified atmosphere with 5% CO₂. Then, 150 µL of the solution was transferred to a 96-well plate, and the fluorescence (Ex/Em = 530/590) was measured on a Synergy^TM HT Multi-Mode Microplate reader (BioTek, Santa Clara, CA, USA). The measured values were corrected for the background control (CCM with resazurin). Each experiment was performed in triplicate.