Anti mouse cd40 pe

For maturation, DCs (2x10⁶ cells/ml) were stimulated as for ELISA and supernatants were discarded. Next, cells were washed twice in PBSAz, incubated with anti-mouse CD16/CD32 BD Fc Block (3 μg/ml; BD Biosciences, San Jose, CA, 553141) for 10 min on ice to block unspecific binding. Subsequently the cells were stained with monoclonal anti-mouse CD40-PE (2μg/ml, 12–0401), CD86(B7-2)-APC (1.25 μg/ml,17–0862) (both from eBiosciences) and CD80(B7-1)-FITC (1.25 μg/ml; BD Pharmingen, San Diego, CA, 553768) for 45 min at RT. Cells were then washed twice in PBSAz, fixed and analyzed by flow cytometry. MFI was used as a measure of maturation based on counting 10,000 cells. To analyze cell viability, the DCs were washed twice in ice-cold DPBS after 1 or 20 h of stimulation with bacteria and their supernatant. Next, the cells were re-suspended in BD Binding buffer, stained with anti-mouse Annexin V-PE and 7-AAD-PerCpCy5.5 (BD Pharmingen, San Diego, CA, 559925) for 15 min at RT and analyzed by flow cytometry based on counting 5,000 cells. Percentages of apoptotic, dead or double positive cells were used. Data analysis was performed using FlowJo Version 10 software (Treestar Inc).

NIH3T3 or MPEK stable cell lines were seeded into a 6-well plate. Some wells received 10 nM rim. Cells were incubated at 37°C in 5% CO₂ for 48–72 hours. Cells were harvested using trypsin, then stained and analyzed for surface expression and upregulation by flow cytometry. NIH3T3 cell lines were stained with anti-mouse-H2-K^q-AlexaFluor 647 (Cat# 115106), anti-mouse-CD80-PerCpCy5.5 (Cat# 104722), anti-mouse-CD86-APC/Cy7 (Cat# 105030) (BioLegend), and anti-mouse-CD40-PE (Cat# 12-0401-82, eBioscience). MPEK cell lines were stained with anti-mouse-anti-H2-K^b-PE (Cat# 116508), anti-mouse-CD80-PerCpCy5.5, anti-mouse-CD86-APC/Cy7, and anti-mouse-CD40-PE.