Bio plex 100 reader

Each PSgc probe was bound to a different carboxylated microsphere as described previously (Chen et al., 2010 (link)). The working microspheres consisted of eight types of beads, and each bead was coupled to a PSgc-specific probe. Hybridization was performed in a 50 μl mixture that included 17 μl of biotin-labeled PCR product and 33 μl of working microspheres, with denaturation at 95°C for 10 min and incubation at 55°C for 17 min in a thermal cycler. The hybridization product was then transferred to a filter plate and washed three times with 1× TMAC buffer at 1,000 rpm for 1 min. For detection, 80 μl of streptavidin-R-phycoerythrin in 1× TMAC buffer was added to each well, followed by incubation at 53°C for 20 min. Finally, the signals for each set of beads were measured using a Bio-plex 100 reader (Bio-Rad) according to the manufacturer’s protocol. Data were analyzed using Bio-plex Manager 4.0, and the data are presented as the median fluorescence intensity (MFI). The cut-off value for a positive result was defined as three times greater than the mean MFI value of the background.

24 hours following transfection, mice were culled and bronchoalveolar lavage fluid collected and processed for cytokine and chemokine quantification as previously described42 (link). The presence of inflammatory cytokines in BALF was assessed and quantified using the Cytokine Mouse 10-Plex Panel (Life Technologies, Paisley, UK) and plates were read using a Bio-Plex 100 reader (Bio-Rad, Hertfordshire, UK) and data collected with Bio-Plex Manager software (v 4.1.1; Bio-Rad, Hertfordshire, UK).