Anti cd40 apc

Spleens were harvested as described above and cell suspensions were infected at a MOI of 5 with RABV, RABV-ED51-mBAFF or equivalent volume of PBS, and incubated for 2 days 37°C and 5% CO₂. Cells were harvested and plated at 10⁶ cells/well of a 96-well plate, pelleted at 300 x g, washed in FACS Buffer (PBS containing 2% FBS). Cells were incubated with Fixable Live/Dead-Aqua (Thermofisher), washed with FACS Buffer and incubated with CD16/32 FcBlock (BD Biosciences). Cells were stained with surface antibody mixture, including (0.2 ug/ml each) anti-B220-PerCP (Clone RA6B2; BD Biosciences), anti-CD40-APC (Clone 1C10; eBiosciences), anti-CD69-V450 (Clone 41:2F3; BD Biosciences), and anti-MHC-II-Alexa Fluor 700 (Clone M5/11415.2; BD Biosciences) for 30 minutes. Cells were fixed in 3% paraformaldehyde (Affimetrex) for 30 minutes, washed, and permeabilized using BD Perm/Wash (554723; BD Biosciences) for anti-Rabies-N-FITC (FujiRebio) intracellular staining. Cells were suspended in FACS buffer and analyzed using LSRII flow cytometer. Data was analyzed using FlowJo Software. To compare two groups of data, an unpaired, two-tailed Student’s t test was use (*p≤0.05; **p≤0.01; ***p≤0.001; N = 3 completed in duplicate).

For in vitro experiments, BMDCs were generated as described previously^{51 (link)}. Cells were plated on day 10 at concentrations of 1 × 10⁶ cells/ml and stimulated 2 h later with decreasing doses of M9 OMVs, 5 ng/ml E. coli LPS (Sigma) or left unstimulated for 24 h. For the analysis of surface marker expression, cells were incubated with AQUA LIVE/DEAD fluorescent dye (0.5 μl) (Invitrogen) for 30 min. Cells were subsequently stained with anti-CD11c (PE-Cy7, eBioscience), anti-CD40 (APC, eBioscience), and anti-CD80 (PerCP-Cy5.5, BD Biosciences) then measured by flow cytometry as described above. The levels of IL-12p70, IL-18, and IL-6 cytokines in culture supernatants were measured by multiplex assay (BioRad).
For in vivo studies, C57Bl/6 mice were injected intraperitoneally with 100 μl saline control or 10 μg OMVs that were pre-labeled with CFSE (CellTrace, Life Technologies) according to the manufacturer’s instructions. After 6 h, cellular exudates were obtained by peritoneal lavage. Cells were stained with anti-CD11c, anti-CD40 (PE), anti-CD80 (PE-CF594), anti-CD86 (BV605), anti-MHC class I (APC), anti-MHC class II (PerCP Cy5.5), and analyzed by flow cytometry. The levels of IL-12p70, IL-18, and IL-6 cytokines in lavage fluid were measured by multiplex assay (BioRad).