Anti cd40 pe cy5

Live cells were discriminated by a live/dead fixable aqua dead cell stain (Molecular Probes). For staining DCs, murine anti-CD11c PE-Cy7 (clone N418, eBioscience), anti-CD11b APC-Cy7 (clone M1/70, Biolegend), anti-CD40 PE-Cy5 (clone 1C10, eBioscience), anti-CD86 APC (clone GL1, eBioscience), and anti–MHC II PE (clone M5/114.15.2, BD) were utilized. For staining T cells, murine anti-CD3 V450 (clone 500A2, BD), anti-CD4 Alexa700 (clone RM4-5, BD), anti-CD8 PerCP (clone 53–6.7, BD), anti-TCR γδ BV605 (clone GL3, Biolegend), anti-CD44 APC-Cy7 (clone IM7, BD), anti-CD45.1 BV785 (clone A20, BioLegend), and anti-CD45.2 BV650 (clone 104, BioLegend) were utilized to stain for surface markers. Murine anti-RORγt PE (clone B2D, eBioscience), anti-TNFa PE-Cy7 (clone MP6-XT22, BD), anti-IFN-γ APC (clone XMG1.2, eBioscience), anti-IL-2 FITC (clone JES6-5H4, BD), and anti-IL-17 PE-CF594 (clone TC11-18H10, BD) were stained intracellularly with the BD Cytofix/Cytoperm or BD Transcription Factor kit as per manufacturer’s instructions. Staining for cell-surface markers was done by resuspending ∼1-2x10⁶ cells in 100 ml PBS with 2% FBS containing the antibody mixture at 4°C for 30 min and then washing with PBS containing 2% FBS. Data were immediately acquired using an LSRII flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (FlowJo LLC, Ashland, OR).

BMDCs of each treatment group in experiment (1) were collected and were divided into two tubes, to which anti-mouse MHCII-PE-(yanin5-conjugated) Cy5, anti-mouse CD80-FITC, and anti-mouse CD11c-PE (phycoerythrin), anti-mouse CD86-FITC, and anti-CD40-PE-cy5 (eBioscience), respectively, were added, followed by the addition of 500 μl of 2% PFA in PBS, pH 7·4. A blank control was included. All samples were incubated in the dark at 4°C for 30 min, and were then subjected to flow-cytometry detection of surface molecules of BMDCs. Data were collected for at least 1×10⁴ cells, and the experiment was carried out four times.