Fitc conjugated anti mouse cd40

After test DCs were incubated with TCL and candidate adjuvants or LPS-treated (see above) DC samples were washed and re-suspended in wash buffer (i.e., PBS supplemented with 1% BSA and 2 mM EDTA) and seeded into a 96-well plate (10⁵ cells/well). For blocking of Fc receptors, DCs were pre-incubated with purified anti-CD16/CD32 antibody (Pharmingen, San Diego, CA) for 15 min at 4°C. Subsequently, test cells were stained with fluoresceinisothiocyanate (FITC)-conjugated anti-mouse CD40, CD80, CD86 and MHC class II, or with phycoerythrin (PE)-conjugated anti-mouse CD11c and MHC-II antibodies (BD Pharmingen, San Diego, CA, USA) for another 30 min at 4°C. Cells were washed twice with wash buffer and resuspended in a 4% paraformaldehyde solution. The phenotypic change of test DCs was analyzed with a BD LSR II flow cytometer and BD FACSDiva software (BD, Franklin Lakes, NJ). The four parameters simultaneously collected were forward scatter (FSC), side scatter (SSC), log FITC and log PE. The gating scope (P9) was gated to exclude debris and aggregated cells on a FSC/SSC histogram (S1B Fig); 10,000 cells were counted in P9 and data were recorded.

Cells were washed twice in cold phosphate buffered saline (PBS) and resuspended at 1 × 10⁶ cells/mL in FACS buffer (PBS/0.1% NaN₃/1% FBS). Cells were blocked with rat anti-mouse CD16/CD32 (BD Pharmingen) at 4°C for 5 min and then stained for 30 min with FITC-conjugated anti-mouse-CD40 and PE-conjugated anti-mouse CD86 (BD Pharmingen) on ice in the dark. For isotype controls, FITC-conjugated rat IgG2a κ or PE-conjugated rat IgG2a κ (BD Pharmingen) was used. The cells were washed twice and resuspended in FACS buffer. Ten thousand cells were collected for each sample and analyzed on a Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA). The data were analyzed with Kaluza software.