Table 1 lists the primers that
were used for mRNA quantification. Samples were analysed using a Bio-Rad iCycler iQ (Bio-Rad, Hercules, CA). Changes in gene expression were determined by calculating the Δ cycle threshold (Ct) by subtracting the Ct for ribosomal protein L19 (RPL19) (reference gene) from the Ct of the gene of interest for each sample.26 The ΔCt of the control was subtracted from the corresponding treated sample giving rise to the ΔΔCt. The fold change was derived from the equation 2−[ΔΔ]Ct. To confirm that the reference gene ribosomal protein L19 was stably expressed in MoDCs and BDCs, a comparison was performed using either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or RPL19 as the selleck products reference gene. Similar trends in fold change were observed. Complementary DNA was diluted to generate
a standard curve whose correlation coefficient was > 0·99. The efficiency of qPCR was determined from the slope using the equation (10[−1/M] − 1) × 100 and ranged between 90% and 110%. To evaluate changes in cytokine secretion, 1 × 106 MoDCs or BDCs were incubated in 1 ml culture medium for 24-hr in six-well plates (Corning) and culture supernatants were collected. Concentrations of IL-6, Lenvatinib mw IL-8 and IL-10 were assayed using commercial kits as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN). The ELISA for IFN-α, TNF-α and IL-12 were performed as previously described.27 Statistical analysis was performed by non-parametric Mann–Whitney U-tests (P-value < 0·05) using the statistical software programme graphpad prism 5 (GraphPad Software, Inc., La Jolla, CA). In this study, 800 ml of EDTA blood yielded approximately 2 × 109 PBMCs. Following CD14+ selection, an average of 2 × 108 monocytes were cultured in the presence of IL-4 and GM-CSF to Terminal deoxynucleotidyl transferase generate MoDCs. On day 6, approximately 2 × 107 MoDCs were harvested and cultured for use. The CD14− population
was positively selected for cells expressing CD172, which equates to the BDC (CD14− CD172+) population. Approximately 3 × 107 BDCs were therefore isolated and rested overnight. In contrast to other studies, the protocol used in this study resulted in lower numbers of MoDCs compared with BDCs from an equal amount of blood.28 Dendritic cell morphology is characterized by a large cytoplasmic cell mass and extrusion of dendrites which increase the surface area available to sample and take up antigens. In this study, the morphologies of Giemsa-stained MoDCs (Fig. 1a) and BDCs (Fig. 1b) were compared. Both DC populations displayed a typical DC morphology, characterized by an irregular cell border with a large cytoplasmic cell mass. Expression of cell surface markers CD172, MHC II, CD16, CD1, CD80/86 and CD14 was assessed by flow cytometry in 6-day-old MoDCs and BDCs (Table 2). Both MoDCs and BDCs expressed all of these markers; however, BDCs showed similar expression of CD172 and MHC II, higher expression of CD16 and lower expression of CD80/86 and CD1.