Plasma samples were collected from ACD-anticoagulated blood and frozen in liquid nitrogen until use. The levels of the cytokines IL-1β, IL-1Ra, IL-8, IFN-γ, IFN-α, IP-10, MCP-1, RANTES, MIP-1β and TNF-a in plasma were measured using a Multiplex cytokine immunoassay (Bio-Plex Human Cytokine Assay). Levels of PF4/CXCL4 and MIF were determined using a standard capture ELISA Kit (R&D Systems) according to manufacturer’s instructions.
Platelets (109 per mL) isolated from 10 dengue-infected and 6 HIV + dengue coinfected patients were incubated at 37 °C in a 5% CO2 atmosphere. After 4 hours of incubation, platelets were pelleted, the supernatants were harvested and the secreted levels of PF-4/CXCL4, RANTES/CCL5 and IL-1β were measured using standard ELISA protocol according to manufacturer’s instructions (R&D systems). PF4/CXCL4, RANTES/CCL5 and IL-1β were also measured in supernatants of platelets obtained from the same patients at the recovery phase.
Platelets (109 per mL) isolated from 10 dengue-infected and 6 HIV + dengue coinfected patients were incubated at 37 °C in a 5% CO2 atmosphere. After 4 hours of incubation, platelets were pelleted, the supernatants were harvested and the secreted levels of PF-4/CXCL4, RANTES/CCL5 and IL-1β were measured using standard ELISA protocol according to manufacturer’s instructions (R&D systems). PF4/CXCL4, RANTES/CCL5 and IL-1β were also measured in supernatants of platelets obtained from the same patients at the recovery phase.