Human inflammatory cytokines multi analyte elisarray

To determine cytokine production by hMDMs, a total of 3 × 10⁶ cells per well were plated in 6 well plates and infected with L. pneumophila at an MOI of 20 for 1h and then treated for 1h with gentamicin to kill remaining extracellular bacteria. Culture supernatants were collected 6h post-infection and then analyzed using the Milliplex (Millipore) assay. Assays were performed according to the manufacturer’s instruction. Standards or culture supernatant samples were mixed with antibody-bound magnetic beads, and incubated overnight at 4 °C. Beads were washed and then incubated with the biotinylated detection antibody for one hour at room temperature. The beads were incubated with phycoerythrin-labeled streptavidin for thirty minutes at room temperature and the median fluorescent intensities were quantified with a Bio-plex 200 analyzer and analyzed with Bio-plex Manager 6.0 software. All samples were measured in duplicate. In some experiments 1mM NaF was added to block glycolysis 1h prior to infection and maintained throughout the experiment. Following, 6 h infection, culture supernatants were retained and immediately analyzed for cytokines using Human Inflammatory Cytokines Multi-Analyte ELISArray™ (Qiagen), following the manufacturer’s instructions. Infections were performed in triplicate.

The levels of pro-inflammatory cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17α, IFN-γ, TNF-α, GM-CSF) were assessed in conditioned media from AMC treated with MALP-2 (100 ng/mL) for 24 hours using the Human Inflammatory Cytokines Multi-Analyte ELISArray (Qiagen, Valencia, CA), as per manufacturer’s instructions. The absorbance reading from the negative control for each cytokine was subtracted from sample absorbance readings to remove background. The resulting data was analyzed as percent fold-change between paired untreated and treated AMC.