For the original untreated negative control iDCs, after

cell transfer to a new 24-well plate, one well still remained untreated whereas Stem Cells inhibitor the other well was treated with LPS. After another 24 hr (Day 2), contents of each well were collected for either cell or cytokine assays. After DC treatment with chemokines (Day 1) and subsequent LPS stimulus (Day 2), cell viability was also determined using Trypan blue exclusion. All treatments and controls exhibited at least 90% viable cells (data not shown). Hereafter, any combination of CCL3 and CCL19 at a specific ratio will adhere to the nomenclature: CCL3 + 19 (ratio). To measure the endocytic capacity of DCs upon chemokine or subsequent LPS treatment, DCs were incubated with fluorescently labelled OVA 24 hr after chemokine treatment (Day 1) or 24 hr after subsequent LPS treatment (Day 2) and the amount of OVA

internalized by DCs was determined using flow cytometry. Immature DCs treated with individual chemokines or chemokine combinations exhibited endocytic capacity comparable to untreated iDCs (Fig. 2a). As expected, upon subsequent LPS maturation, iDCs treated only with LPS reduced their www.selleckchem.com/products/ink128.html endocytic capacity significantly compared with untreated iDCs. However, iDCs pre-treated for 24 hr (Day 1) with individual chemokines or an equal Obatoclax Mesylate (GX15-070) combination of CCL3 + 19 (5 : 5), then subsequently treated with LPS exhibited an endocytic capacity similar to untreated iDCs. Surprisingly, even after subsequent

LPS treatment, iDCs pre-treated with CCL3 + 19 (7 : 3) showed an endocytic capacity 36% higher than untreated iDCs, whereas iDCs pre-treated with CCL3 + 19 (3 : 7) exhibited a 30% lower endocytic capacity than untreated iDCs (Fig. 2a,b). When endocytic capacity (MFIs by flow cytometry) was recalculated, now normalized to the value of endocytic capacity for untreated iDCs on Day 1, iDCs pre-treated with CCL3 + 19 (7 : 3) retained 57% of their endocytic capacity, even after subsequent LPS treatment. Conversely, the normalized endocytic capacity of untreated iDCs or iDCs treated with only LPS was reduced to 44% or 15%, respectively (Fig. 2c). Even though there is no direct evidence explaining why the endocytic capacity of untreated iDCs decreased over time, this natural decrease was presumably attributed to effects of the GM-CSF in the culture media[41-43] and of the cell transfer (Fig. 1)[41] on minimal maturation of these DCs during the 3-day culture in this study.