The MFI of the ice control cells was subtracted from that of cells incubated at 37° with OVA per treatment or control. Data were analysed using the FlowJo Software (Tree Star). Endocytic behaviour and morphology of DCs treated with chemokines and/or subsequent LPS were examined by confocal laser scanning microscopy. Briefly, DCs were collected on Day 1 and Day 2 post-treatment and resuspended in medium (without phenol red) at 1 × 106 cells/ml. Then, each sample was incubated with 5·8 μg/ml of

fluorescent Alexa Fluor 488-Ovalbumin (OVA) (a model antigen) (Invitrogen) or 0·5 mg/ml Lucifer Yellow (LY) (Invitrogen) for 30 min at 37°. OVA is known to be internalized by DCs by a combination of receptor-mediated endocytosis and fluid-phase macropinocytosis[17] whereas Idelalisib manufacturer LY is internalized by only fluid-phase macropinocytosis.[34] RAD001 mouse After incubation,

any excess fluorochrome bound to cell surfaces was quenched for 3–4 min on ice using 0·5% Trypan Blue/2% FBS/1× PBS solution. After two sequential quenching steps, cells were washed three times using 1% BSA/PBS solution, resuspended in complete medium (without phenol red) at 1 × 106 cells/ml, then the cell suspension was used to submerge a glass cover slip and allowed to incubate for 4 hr at 37° to induce cell attachment to the cover glass. After incubation and another washing, cells were fixed with 2% paraformaldehyde for 10 min at room temperature, and permeabilized with 0·05% Triton-X 100 (Sigma) for 15 min at room temperature. Then, cells were washed three times, and incubated with Adenosine Texas red-X phalloidin (Invitrogen) at 0·165 μm in 1% BSA/PBS solution for 20 min at room temperature. Cells were then washed and permanently mounted using Fluoromount G (SouthernBiotech, Birmingham, AL). Microscopic images were acquired with a Zeiss 510 META confocal laser scanning microscope (Zeiss, Thornwood, NY) using 100× /1·4 NA oil objective. For this analysis, at least seven cells were examined per treatment condition. Each cell was ‘optically sectioned’ by collecting x–y plane images or slices

at 12–14 different z-direction altitudes through the cell (x-y slices were collected every Δz = 507 nm). A single x-y slice was selected from the middle of the z-stack of images (middle of the cell) for reporting here. To measure expression levels of DC surface markers, cells were resuspended in FACS buffer, blocked with anti-mouse Fcγ III/II receptor monoclonal antibody (clone 2.4G2; IgG2bκ) (BD Pharmingen), and stained with saturating concentrations of fluorescently conjugated rat or mouse anti-mouse monoclonal antibodies against CD86 (clone GL1; IgG2aκ), MHC Class I (H-2Kb) (clone AF6-88.5; IgG2aκ) and MHC Class II (I-2Ab) (clone AF6-120.1; IgG2aκ) (all from BD Pharmingen) for 30 min at 4° in the dark. After staining, cells were extensively washed three times using ice-cold FACS buffer and then, analysed immediately with 10 000 events per sample using FACS Canto (BD Biosciences).