Alexa fluor 647 mouse anti iκbα

Flow cytometry analysis was used to investigate the expression of signaling pathway markers. Briefly, cells were seeded in 24 well plate at 0.5 × 10⁵ cells/ml in serum free media overnight. Cells were treated with nSMase inhibitor GW4869 or DMSO (vehicle) then subjected to stimulation with TNF-α (10 ng/ml) or BSA (vehicle) for 10 min. After stimulation, cells were collected and washed. Cells were then incubated with fixation/permeabilization buffer (cat# 00-5523-00, eBioscience, San Diego, CA, USA) for 20 min in 4 °C, followed by washing and staining with mouse anti-human p-NF-kB P65-PE (20ul/test; cat # 558423; BD Biosciences) and Alexa Fluor 647 mouse anti-IκBα (5ul/test; cat # 560817; BD Biosciences) or control isotypes (mouse IgG2b, κ-PE, Cat# 559529; Mouse IgG1-Alexa Fluor 647, Cat # 560817) for 30 min. The cells were then washed and resuspended in PBS supplemented with 2% FCS for FACS analysis (FACSCanto II; BD Bioscience, San Jose, USA). FACS data analysis was performed using BD FACSDiva Software 8 (BD Biosciences, San Jose, USA). We used unstimulated cells as controls for different treatments.