Mouse igg1 alexa fluor 647

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.

Cells were detached using the trypsin-free Accutase Solution (Sigma), resuspended in culture medium containing 0,1% azide and a 1:100 dilution of mouse or rat immunoglobulins (Sigma) as blocking agents, and incubated for 30 min on ice. The specific antibodies were then added to the cell suspension at a 1:100 dilution, and an additional incubation was carried out on ice for 30 min. Cells were then washed repeatedly. All specific antibodies were purchased from BD: anti-mouse-CD11b-BB515 (rat, BD 564454), anti-mouse-F4/80-Alexa Fluor 647 (rat, BD 565853), anti-human-IL-10RA-Alexa Fluor 647 (rat, BD565255), anti-human-IL-10RB-Alexa Fluor 647 (mouse, BD 564372). Control antibodies were: mouse IgG1-Alexa Fluor 647 (BD 557714) and rat IgG2b-BB515 (BD 564421). Cells were analyzed in an Accuri C6 (BD BioSciences) cytometer running on version 1.0.264.21 of the Accuri C6 software. Details on the gating and quantification strategies are provided in the figure legends and Supplementary Fig. 17.