Bio plex multiplex cytokine assay kit

Plasmas were frozen at −20°C until use. IFN‐γ, TNF‐α, IL‐1β, IL‐8, IL‐4, and IL‐10 were measured by a Bio‐Plex multiplex cytokine assay kit according to manufacturer's instructions (Bio‐Rad Labs, Hercules, CA, USA). In brief, plasmas were centrifuged at 10,000g for 10 min at 4°C to remove precipitates and platelets. Bead‐coupled capture antibodies were added to each well of the plate, incubated for 10 min, and washed twice. Fifty microliters of standards and plasma were added to the wells, the plate is incubated, and shaken gently for 30 min at room temperature in the dark. After three washings, a biotinylated detection antibody was added to the wells, and the plates were incubated for 30 min. Finally, PE‐labeled streptavidin was added to the wells and incubated for 10 min. The plate was read on a Bio‐Plex Luminex 200 system. The different cytokine concentrations were calculated in picogram/milliliter using Bio‐Plex Manager version 6.0 software (Bio‐Rad Labs, Hercules, CA, USA). Standard curves for each cytokine were made by means of standards provided by the manufacturer.

The Bio‐Plex multiplex cytokine assay kit (Bio‐Rad) was used to investigate the effects of GW agent exposure on cytokine and chemokine profiles of GW agent treated (n = 14) and control (n = 14) mouse plasma and brain homogenate samples (plasma samples, 1:4 dilution using Bio‐Plex sample diluent; tissue lysates, 1:2 dilution of lysates at 900 μg/mL of protein in Bio‐Plex sample diluent). The Bio‐Plex Pro™ Mouse Cytokine Th17 Panel A kit (Bio‐Rad) and the Singleplex Interleukin 4 kit (Bio‐Rad and Invitrogen, MD) were used in this study to assess tumor necrosis family‐α (TNF‐α), interferon‐γ, interleukin 6, interleukin 1β (IL‐1β), interleukin 10 (IL‐10), interleukin 17 (IL‐17), and interleukin 4 levels in plasma and brain homogenates, at 5 months postexposure.