Plate based autosampler

The HT-29 human colonic epithelial cell line from American Type Culture Collection (ATCC, Manassas, VA, USA) was used. Cells were propagated in McCoy's 5A medium containing 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mm l-glutamine (Gibco Life Techologies). Cells were trypsinized from flasks, washed in fresh medium, and resuspended in 96-well assay plates (Costar Corning) at a density of 3 × 10⁵ cells per well. Oclacitinib (0.0015–30 μm)) or vehicle control was added to cells while on ice for 30 min. One nanogram per milliliter hIL-13 (R&D Systems) was then added. Cells were incubated in a 37 °C water bath for 30 min then fixed in 1.75% formaldehyde in PBS, washed in PBS containing 0.5% BSA, and incubated overnight at −20 °C in absolute methanol. Fixed cells were stained with PE-labeled antibody to human pSTAT6 (pY641; BD Biosciences, San Jose, CA, USA). Samples were analyzed with an FACSCalibur equipped with a plate-based autosampler (BD Biosciences) and analyzed using FlowJo software, version 7.6.1 (Tree Star Inc., Ashland, OR, USA). Data were expressed as mean fluorescence and then expressed as percent control. Dose–response data were then analyzed using a 4-parameter logistic equation.