Bio plex 100 array reader

Within four hours of collection, the aliquots of AH and serum were frozen at -20°C and stored at this temperature for up to two months, thereafter at -80°C until the time of analysis, which was conducted simultaneously for all samples.
The Bio-Plex® multiplex immunoassay beads system (Bio-Plex 100 array reader and Bio-Plex Manager software, version 6.1, Bio-Rad, Hercules, CA, USA) was used to simultaneously quantify the concentrations of 43 cytokines and chemokines according to the manufacturer instructions, as previously described [18 (link)]. A concentration standard was run in parallel on each test plate. It represented the average of triplicate standard dilutions of each corresponding chemokine/cytokine. A standard curve was generated and the sample concentrations were determined by curve-fitting. The assays were performed in a blinded manner by an experienced technician [29 (link)].

The cytokine analyses were performed using the Bio-Plex® multiplex beads system (Bio-Plex 100 array reader with Bio-Plex Manager software version 6.1, Bio-Rad, Hercules, California). With this system, it is possible to quantify in parallel the concentration of numerous cytokines and chemokines in a single sample with small volume. In the present study, the levels of 40 pro- and anti-inflammatory chemo- and cytokines were quantified in each sample of aqueous humor. To this end, fluorescently-labelled magnetic microspheres were coupled to specific capture antibodies and mixed with the samples in which the cytokine concentrations were unknown. Biotinylated detection antibodies and streptavidin R-phycoerythrin were introduced into the mixture, which was then analyzed by flow cytometry. Two lasers identified the microsphere type and quantified the amount of the bound antigen. The readings were compared with the values on a standard curve, which represented the average of triplicate standard dilutions of the corresponding cytokines that were run on the same plates. All measurements were performed in a blinded manner by a laboratory technician who was experienced in executing the technique.