Then sequential treatments of these prepared JAWS II iDCs and examination of them were performed as described in the Results section. The effects on DCs of chemokine pre-treatment followed by LPS stimulus (to initiate

maturation) were assessed by measuring levels of endocytic ability. To quantify endocytic ability, DCs collected on Day 1 (24 hr after no treatment or the described chemokine treatment) and on Day 2 (24 hr after subsequent LPS treatment) were resuspended in medium (without phenol red) at 1 × 106 cells/ml. Then, each sample received 3·33 μg/ml fluorescent Alexa Fluor 488-ovalbumin (OVA) (a model antigen) (Invitrogen) for 30 min at 37°. After incubation, Quizartinib nmr any excess fluorochrome bound to the cell surface BAY 73-4506 mw was

quenched for 3–4 min on ice using a 0·5% Trypan Blue/2% FBS/1× PBS solution. After two repetitive quenching steps, cells were thoroughly washed using ice-cold FACS buffer (2% FBS/1× PBS) and then immediately examined using a FACS Canto (BD Biosciences, San Jose, CA). Negative control DCs were separately prepared by incubation of DCs with the model antigen on ice. The mean fluorescence intensity (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 Inc., Ashland, OR). The model antigen (OVA) degradation (processing) by DCs was also examined using flow cytometry. Here, DCs were treated with BODIPY-conjugated DQ-OVA (Molecular Probes/Invitrogen), 4��8C a self-quenched conjugate of OVA that exhibits

bright green fluorescence only upon proteolytic cleavage releasing the dye molecule from the OVA. To quantify antigen degradation kinetics, this assay was carried out at 30 min, 1 hr and 2 hr after OVA incubation. DQ-OVA was applied at the concentration identical to the OVA of the antigen uptake assay above. Briefly, after DCs were collected on Day 1 and Day 2, DCs from control or sample wells were divided into three groups and resuspended in medium (without phenol red) at 1 × 106 cells/ml per group. Then, each group was incubated with 3·33 μg/ml of DQ-OVA for 30 min, 1 hr, or 2 hr at 37°. After each time-point, cells were extensively washed using PBS and then fixed with 2% paraformaldehyde (diluted from Cytofix; BD Pharmingen, San Jose, CA) for 10 min at room temperature. Fixed cells were washed twice using ice-cold FACS buffer, then examined using a FACS Canto (BD Biosciences).