Synergy h1 plate reader

The 2,2-Diphenyl-1-picrylhydrazyl (DPPH)-free-radical scavenging activity was measured according to the method described by Schaich et al. [31 (link)], with modifications for a 96-well microplate scale. The stock solution (600 μM) was prepared in methanol and stored in the dark at −20 °C. A daily DPPH solution was prepared by dilution with methanol to achieve an absorbance of 0.600 ± 0.100 at 515 nm. For the assay, 25 μL of sample (biocompatible concentrations of 0.12 g/L for the delivery system and 0.005 g/L (15 µM) for curcumin, determined in our previous work [21 (link)]), Trolox (7.5–240 μM), ascorbic acid (0.05 g/L, 280 μM) or solvent were added to 175 μL DPPH daily solution. The mixture was incubated for 30 min at 25 °C and the absorbance was measured at 515 nm with a Synergy H1 plate reader ( Agilent, Santa Clara, CA, USA). The scavenging activity was expressed as percentage (%) reduction in absorbance related to the control. Regression equations between DPPH scavenging and Trolox concentration were calculated, and the results expressed as µmol TE (Trolox equivalent) per gram of sample. The DPPH scavenging effect percentage was determined according to Equation (4): $$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\left(\mathrm{\%}\right)=\left[\frac{A_{control}-A_{sample}}{A_{control}}\right]\times 100$$
where A_control and A_sample are the absorbances at 515 nm of control and sample, respectively.

A549 cells were seeded in phenol red-free DMEM with 10% FBS the day before infection. BlaM-Vpr containing HIV-1 particles (50 pg p24) pseudotyped with wild-type or GPc were bound to cells by centrifugation at 1550xg for 30 min, 4°C. Cells were washed with cold phenol red-free DMEM with 10% FBS and 20 mM HEPES (GE Healthcare Life Sciences) to remove unbound viruses and incubated at 37°C to initiate virus-cell fusion. Cells were then placed on ice, loaded with the CCF4-AM BlaM substrate (Life Technologies), and incubated at 11°C overnight to allow substrate cleavage. The cytoplasmic BlaM activity (ratio of blue to green fluorescence) was measured using a Synergy H1 plate reader (Agilent Technologies, Santa Clara, CA, USA).

Quantification of cytochrome-C released in response to AEA treatment in BAEC challenged with LPS was carried out using the Promega Cytochrome-C Assay (Promega, Madison, WI, USA). Briefly, BAEC were cultured in white 96-well plates following the experimental design and the assay was carried out following manufacturer’s recommendations. Luminescence was read on a BioTek Synergy H1 plate reader (Agilent, Santa Clara, CA, USA).