Synergy 2

Nitric oxide was analyzed from frozen plasma samples using a nitric oxide assay kit (Invitrogen) according to the manufacturer’s instructions; the nitrite concentration was calculated based on standard curve measuring optical density at 540 nm on a plate reader (BioTek Synergy2). Corticosterone was measured colorimetrically in plasma using DetectX corticosterone enzyme immunoassay kit K014-H1 (Arbor Assays). After incubation, the reaction was read at 450 nm on a BioTek Synergy2 plate reader.

Cells were seeded in 96-well plates with a 5000 cells/well density. After the treatment, 10 μL of alamarBlue reagent (ThermoFisher, MA, USA) was added to each well, mixed and then incubated for 4 h at 37 °C in a CO₂ incubator. The absorbance of each sample was measured using a scanning microplate spectrophotometer reader (Synergy 2, Biotek, Winooski, VT, USA) by absorbance detection at 570 nm or fluorescence detection at excitation and emission wavelengths of 540–570 and 580–610 nm, respectively. IC50 values for each treatment were calculated using the GraphPad software. For cristal violet proliferation assay, cells were seeded in 96-well plates with a 5000 cells/well density. Twenty-four hours later, cells were treated or not with 0.5 µM epi-enprioline or DMSO for up to 72 h. Cells were washed once with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min. The PFA was removed, and cells were stained for 30 min at room temperature with an aqueous solution containing 0.1% (w/v) crystal violet. Cells were washed three times with distilled water before the administration of 200 µL/well of 10% acetic acid and shaking with micropipettes. The absorbance of each sample was measured using a scanning microplate spectrophotometer reader (Synergy 2, Biotek) by absorbance detection at 595 nm.

The DPPH assay was conducted after optimizing the previously described protocol [41 (link)]. Briefly, 100 µL of resveratrol-loaded nanogel dispersion (78 µg/mL) and a hydroalcoholic solution of pure resveratrol in the same concentration were added to a 100 µL ethanol solution of DPPH (440 µg/mL). Immediately after that, the absorbance of the reduced form of DPPH was measured in a multiplate reader, Synergy 2 (BioTek Instruments, Inc., Highland Park, Winooski, VT, USA), at 517 nm and 25 °C.
The ABTS assay was performed after modification of previously described procedures [42 (link),43 (link)]. A stock solution of ABTS was mixed with potassium persulfate in water, and the mixture was incubated in a dark place at 25 °C for 16 h. Therefore, 100 µL of resveratrol-loaded nanogel dispersion (78 µg/mL) or a hydroalcoholic solution of pure resveratrol in the same concentration were added to 100 µL of a 5% alcoholic solution of ABTS. The absorbance of the samples was measured in a multiplate reader, Synergy 2 (BioTek Instruments, Inc., Highland Park, Winooski, VT, USA), at 734 nm and 30 °C.

A white 96 well plate was labeled in triplicate with the cell line and number of cells plated. Initially, a variety of cell concentrations ranging from 500 to 3000 cells per well were plated to determine the best linear growth rate concentration. The appropriate amount of media was added to cell samples to reach a concentration of 100 μL needed per well. The plate was incubated for 2.5 h at 37 °C. Ten μL of the substrate and 10 μL of enzyme from the RealTime Glo Viability Assay (Promega, Madison, WI, USA) were mixed into 4980 μL of media to make a 2X solution. The solution was placed into a hot water bath for 5 min. Media was removed from the 96 well plate and 50 μL of new media was added to wells. Fifty microliters of substrate-enzyme solution was added to each well. The plate was incubated for 1 h at 37 °C. The Biotek Synergy 2 plate reader was prewarmed to 37 °C before the first reading. The plate was read on the prewarmed fluorescent plate reader (Biotek Synergy 2, Winooski, VT, USA) at 24, 48, and 72 h (488 nm) post plating. Results were graphed to determine linearity of concentrations. The following formula was used to determine the doubling time for each cell line:
(t2 - t1)/3.32 x (log n2 - log n1) where t is time and n number of cells (27) {Hayflick, 1973 #8}.

In this study, we used MTT assay protocol to test whether SF-E alter the oxido-reductase activity in live neurons. Briefly, neurons were treated with specified concentrations of SF-E for 24 h. After incubation, the medium was removed and 100 µL of MTT reagent (0.5 mg/mL) dissolved in DMEM was added to each well. The plates were incubated for 3 h at 37°C, and the formazan particles formed by reduction of the tetrazolium dye to its insoluble formazan were dissolved with 100 µL DMSO in each well, and absorbance at 540 nm was measured with a microplate reader (Biotek Synergy 2, Winooski, VT).
For microglial cells, we used a protocol containing WST1 (Water Soluble Tetrazolium salt) (Clontech, Mountain View, CA) to determine effects of SF-E on cell viability. WST1 has advantage over MTT because it yields a water soluble formazan and can be read directly without the solubilization procedure. Briefly, cells treated with specified concentrations of SF-E were incubated for 16 h. After incubation, the medium was removed and 10 µL of WST1 reagent (1∶10 of premixed reagent) dissolved in 90 µL DMEM was added to each well. The plates were incubated for 30 min at 37°C and absorbance of formazan produced by the dehydrogenase activity of the living cells was measured at 450 nm using a microplate reader (Biotek Synergy 2, Winooski, VT).