Cytokine Array

HeLa, HCjE, HVEC, HFK-2, and HCK were seeded at 10⁶/ml into 6-well plates. THP-1 cells were seeded into 6-well plates at 10⁶/ml into 6-well plates and were differentiated into macrophages using 100 nM PMA. Cells were infected at a MOI of 1 with C. trachomatis 434/Bu, D/UW-3/CX, or A/HAR-13 using complete RPMI 1640 media. The inoculum was spun onto the cells at 900 x g for 15 min. Following centrifugation, the supernatant was removed and replaced with fresh RPMI media. When required, 50 μg/ml of chloramphenicol was added at 1 h post-infection. Infected cells were incubated at 37°C with 5% CO₂ for 24 or 48 h. Supernatants and lysates were collected from each cell type and combined as many cytokines are rapidly secreted. Samples were assayed in duplicate for expression of 36 cytokines using the human cytokine array proteome profiler array following the manufacturers guidelines (R&D systems ARY005B). The cytokine array proteome profiler utilizes a dot blot with capture antibody pre-conjugated to the membrane for relative measurement of cytokines. To control for non-specific binding, each array contained both positive and negative controls. After the experimental procedure, array dot blots were exposed to X-ray film and developed. Image J was used to quantitate spot intensity for arrays. Measurements were acquired by auto-correcting for background based on the negative control spot and measuring the integrated density of each spot. For each array, a circle corresponding to the approximate size of the largest spot on the array (usually the reference spot) was made. This same circle was used to measure every spot on the blot without varying the size. Each array came with internal references spotted at different locations on the blot and present in sextuplet. For each array, the average integrated density of the reference was calculated as well as integrated densities for each spot on the array corresponding to the various cytokines. Each set of samples was averaged and a standard deviation between the duplicate samples was derived. The quotient of the integrated reference density and the cytokine specific spot density was used to represent the relative percentage of the specific spot density as a percentage of the reference density. This allowed control for slight variations in exposure time for different array dot blots. Although uncommon, some spots showed greater integrated density than standards, resulting in values in excess of 100%.