The mice were euthanized by cervical dislocation, in which the neck of each mouse was grasped firmly in one hand and the other hand was used to sharply pull at the base of the tail. Prior to its placement in the glass cabinet for manipulation, the mouse model was doused with 70% alcohol. A small cut was made in the abdominal area with a pair of sterile scissors, just above the mid-line. This was then followed by the removal of the membrane layer to reveal the organs in the mid-section of the body. The femurs of the mice were dislodged and submerged in PBS (phosphate buffer saline) supplemented with antibiotic-mycotic (Sigma-Aldrich, St Louis, MO).

Bone marrow cells were used to isolate mononuclear cells by centrifugation in a Ficoll gradient. All obtained mononuclear cells were maintained at 37°C with 5% CO2. We added IL-4 to the culture medium, which contains GM-CSF for culturing DCs, because a combination of GM-CSF and IL-4 is better able to stimulate mixed leukocyte reactions. The culture medium that was used for all of the experiments was RPMI-1640 (Sigma-Alrich, St Louis, MO) supplemented with 2 mM/L glutamine, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). To produce immature DCs (iDCs), adherent cells were cultured for 6 days in medium containing recombinant GM-CSF and IL-4 at concentrations of 20 ng/mL each. At days 7–12, these cells matured with TNF-α (20 ng/mL), a different cytokine that was added to the complete media. At day 12, mature DCs (mDCs) were confirmed to have DC phenotypes through flow cytometry detection of CD14 (for monocytes), CD40, D80, and CD86 (for DCs).

The phagocytic capacities of iDCs and mDCs were analyzed according to a previously published protocol Phuc et al., 2011. Briefly, 5×10⁴ cells were incubated for 1 h with dextran-FITC (1 mg/mL; Sigma-Aldrich, St Louis, MO) in 100 μL of culture medium at 37°C and 4°C as negative control. Cells were then washed with cold PBS supplemented with 1% bovine serum albumin (BSA) four times before flow cytometry analysis. Phagocytic ability of DCs was confirmed by the appearance of cell populations that expressed FITC fluorescent signals.

Stimulation of CD4⁺ T lymphocyte proliferation was evaluated following the previously published protocol Phuc et al., 2011. The assessment was performed in different groups with the ratio of DC/lymphocytes as follows: 0.25:100, 0.5:100, 1:100, 2:100, and 8:100. The control groups included DC+PHA (phytohemagglutinin, Sigma-Aldrich, St Louis, MO), PHA, and PHA+lymphocytes. PHA concentration was 50 mg/L, and the experiment was conducted on 96-well plates (Nunc, Roskilde, Denmark) and repeated four times. CD4⁺ T lymphocyte proliferation was measured by MTT assay using the Cell Growth Determination Kit, MTT based (Sigma-Aldrich, St Louis, MO). Twenty microliters of MTT was added into each well of the 96- well plates, followed by incubation for 4 h and addition of 150 μL of DMSO. Plates were then thoroughly mixed for 10 min until the crystals completely dissolved. Absorption values (A-values) for each well were measured at a wavelength of 490 nm using micro-plate reader DTX 880 (Beckman Coulter, Brea, CA). An offset value of A and absorption value of a control group (DC+PHA) reflect lymphocyte proliferation. An offset value of absorption in the lymphocyte+ PHA and PHA groups showed proliferation of lymphocytes in the control group. All results were analyzed by Staraphic 7.0 software.

To detect the secretion of cytokines, monocytes were further induced with TNF-α after induction with GMCSF and IL-4, as described above, in culture medium in 24-well plates for 24 h. Supernatant was collected and frozen at −80°C until analysis. The quantity of cytokine IL-12 in the supernatant was determined by ELISA (Abcam, Cambridge, UK) and read on a DTX 880 (Beckman Coulter, Brea, CA).

We observed that monocytes formed small groups similar to the culture of monocytes from human bone marrow Bai et al., 2002, peripheral blood Ratta et al., 1998 or umbilical cord blood Liu et al., 2002Shi et al., 2005 as well as to those observed in some previous studies addressing bone marrow-derived dendritic cells Lutz et al.,1999Lutz and Rossner, 2007Lutz et al., 2000Scheicher et al., 1992. Most of the cells had expanded cytoplasm and small dendritelike structures. Morphologically, the differentiated cells had some characteristics of DCs. These cells had relatively uniform shapes with large heterogeneity of nuclei, many mitochondria and vacuoles, and relatively few particles in the cytoplasm. Cell shape is comparable to DCs ( Figure 1 ).

The results of DC marker analysis are presented in Figure 2 . It was observed that monocytes expressed CD14 but did not express CD40, CD80, and CD86 before induction; they expressed CD86 and CD40 but did not express CD14 and CD80 after induction of iDCs; and they expressed CD40, CD80, and CD86 but did not express CD14 after induction of mDCs.

Antigen phagocytosis

To evaluate the function of DCs, we measured the phagocytic ability of DCs in vitro. Phagocytic activity was assessed by measuring the ability of cells to consume dextran-FITC. Our data showed that monocytes after induction increased their phagocytic ability to 92.28 ± 9.25% in iDCs and 78.54 ± 11.33% in mDCs (p<0.05). Meanwhile, phagocytic ability was only 8.11 ± 3.13% (n = 3) in the group of cells that were cultured in the medium without GM-CSF, IL-4, and TNF-α. In addition, cells that were cultured at 4°C could not engulf the dextran-FITC ( Figure 3 ).

Induced monocyte-stimulated T lymphocytes

The expression of CD80 and CD86 on the surface of the DCs was associated with the activity of activated T lymphocytes. The results showed that induced DCs by monocytes with the cytokines GM-CSF, IL-4, and TNF-α triggered T cell proliferation. Figure 4 reveals that the activity of DCs was significantly different compared with that of the control group (p<0.05) and was augmented when DC concentrations increased (p<0.05).

A-values ( Figure 4 ) are offset of OD values that were measured in the lymphocyte+PHA control group and experimental groups. Because PHA is the strongest stimulant of lymphocytes, the OD value of the control sample was the highest. Therefore, the A-value was smaller when the growth capacity of experimental cells was higher. In a similar manner, the A-value was larger if the stimulation by DC was lower. The experimental results also revealed that DCs differentiated from bone marrow monocytes had the ability to stimulate lymphocyte cells in vitro. This ability depended on the ratio of mixing between DCs and lymphocyte cells; the more DCs added, more lymphocytes were stimulated. Activity of stimulating lymphocyte proliferation is a critical characteristic of DCs in vivo. This feature helps enhance immunity in cancer treatment, which is a key point for success of this approach.

Induced monocytes produced IL-12

To determine the mechanism of activated T cell proliferation, the secretion of IL-12 was evaluated after induction because IL-12 is the most important interleukin in the activation of T lymphocytes. In fact, DCs always interact with other cells in the body. This interaction can occur directly by cell-cell communication via the interaction of surface proteins (as described above) through B7 (CD80 or CD86) with CD28 on lymphocytes; alternatively, this interaction can occur indirectly over a greater distance through cytokines. Typically, DCs produce IL-12 after induction with antigens. IL-12 is a signal that induces CD4⁺ T cells into the Th1 phenotype. Then, it activates the immune system to attack antigens that are presented by DCs.

IL-12 quantity was analyzed by ELISA ( Figure 5 ) and it was shown that there was a statistically significant difference (p<0.5) between monocytes with and without cytokine treatment.