The conditioned media was thereafter collected, centrifuged at 800× g for 30 min, aliquoted and frozen at −80°C prior to evaluation of the levels of human IFN-γ, IL-4, IL-6, LIGHT, MIP-1α also known as CCl-3, TNF-α and VEGF Humann Quantikine Kits, R&D Systems Europe, Abingdon, UK) according to the manufacturer protocol.
For light-grown studies, plants were grown in 16-h light/8-h dark cycling conditions for 14 days on sucrose-containing (1%) media and were thereafter transferred to fresh media containing either no sucrose or 1% sucrose and placed in the dark or white light (70 μEin m-2s-1) for an additional three hours.
Thereafter, media were removed and cells were maintained in GRACE's medium containing 10% FBS.
Thereafter, conditioned media were collected to assess LOX secretion.
After 24 hours, the efficiency of transfection was tested by fluorescent microscopy of green fluorescent protein (GFP), and thereafter the media were changed.
Thereafter, the media were changed for fresh ones supplemented with 100 μ M sodium arachidonate (Sigma-Aldrich) and containing tetradecanoyl phorbol acetate (TPA) 10 n M (Sigma-Aldrich), melatonin 1 n M or the diluent of these drugs (ethanol, at a final concentration lower than 0.0001% per plate).
The media were changed after 5 days, and thereafter every second day for 21 days.
After two weeks, culture media were replaced with fresh ones and thereafter changed every 7 days.
Thereafter, cell culture media were changed and cells were treated with indicated concentrations of CAERS and/or CFEZO extract(s).
The isolated DCs were incubated with the isolated EVs in 48-well plates for 6 h; thereafter, the conditioned media were harvested, and then the levels of IL-12p70 and IL-6 were quantified.
