Rep qbs

HEK-Blue IL-1β cells (hkb-il1bv2, InvivoGen, San Diego, CA, USA) were seeded at 150 μL per 96-well plate overnight, then incubated with 50 μL UV-inactivated conditioned media for another day. UV inactivation was done by using the GS Gene Linker UV Chamber (Bio-Rad, Hercules, CA, USA) following the program setting of sterilization application. For the confirmation of virus inactivation in the experiments, a parallel experiment was done side by side stained with ZIKV viral protein to ensure no virus was alive. HEK-Blue IL-1β cells sense bioactive IL-1β in conditioned media and then activate nuclear factor κB (NF-κB)/AP-1, leading to the production of secreted embryonic alkaline phosphatase (SEAP). The cell culture supernatant was transferred to a flat-bottom 96-well plate and mixed with 150 μL/well QUANTI-Blue Solution, a SEAP detection medium (rep-qbs, InvivoGen). After incubation, the bioactive IL-1β was represented by measuring the SEAP levels by spectrophotometry at 650 nm.

QUANTI-Blue^TM (rep-qbs, InvivoGen, Toulouse, France) is a colorimetric enzyme assay developed to detect SEAP in cell supernatant. The supernatant of unstimulated, stimulated, neutralized, and immunomodulated HEK-Blue^TM cell cultures were collected for SEAP quantification by QUANTI-Blue^TM assay. The measurements were carried out in 96-well plates, and SEAP levels were quantified using a spectrophotometer (Synergy 2, BioTek, USA) at a 655 nm wavelength, according to the provided instructions.