Phospho p38 mapk apc

Activation-induced changes in signaling mediators downstream of TLR4 were measured using flow cytometry. Briefly, 10⁶ UCBMC (n=8/group) were stimulated either for 30 min or 2 hr with 1 μg/mL LPS or left untreated in a 37 °C incubator with 5% CO2. Cells were washed with FACS buffer and surface stained for CD14 and HLA-DR in FACS tubes. Pellets were washed in FACS buffer and resuspended in 100 μL prewarmed PBS (Ca + Mg + free). Cells were fixed immediately by the addition of equal volumes of prewarmed Cytofix Buffer (BD Biosciences, Brea, CA) and thorough mixing and incubating at 37 °C for 10 min. Cells were then centrifuged at 600 g for 8 min. Supernatants were removed leaving no more than 50 μL residual volume. Cells were then permeabilized by the addition of 1 mL 1 X BD PermWash Buffer I, mixed well, and incubated at room temperature for 30 min. Pellets were then washed and stained intranuclearly with antibodies against NF-kB p65 pS529 AF647 (Clone K10-895.12.50, Cell Signaling Technology, Danvers, MA) (for 30 min stimulation samples) or IkBa PE (Clone MFRDTRK, eBioscience, San Diego CA) and Phospho-p38 MAPK-APC (Clone 4NIT4KK, eBioscience, San Diego CA) for 1 hr at room temperature in the dark. Samples were washed twice in PermWash Buffer I, resuspended in FACS buffer, and analyzed on Attune NxT flow cytometer.