Alexa fluor 647 mouse anti stat3 py705

HT-29 cells or EC4 cells were plated in 96-well plates and stimulated with WT or mutant IL-22 for 20 min at 37°C, followed by fixation with paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature. The cells were permeabilized for intracellular staining by treatment with ice cold methanol (Fisher) for 30 min at −20°C. The cells were then incubated with the desired antibodies at a 1:50 dilution for 1 hour at room temperature in autoMACS buffer (Miltenyi). The background fluorescence of the unstimulated samples was subtracted from all samples. Data was acquired using CytoFlex, flow cytometer instrument (Beckman Coulter). The MFI values were background subtracted and represented as a percent of the maximal WT IL-22 value within each experiment and plotted in Prism 8 (GraphPad). The dose-response curves were generated using the “sigmoidal dose-response” analysis. Alexa Fluor 488 Mouse Anti-Stat1 (pY701) and Alexa Fluor 647 Mouse Anti-Stat3 (pY705) antibodies were purchased from BD Biosciences.