TY - JOUR
T1 - Culture and identification of mouse myeloid semimature dendritic cells
AU - Fu, Bi Mang
AU - He, Xiao Shun
AU - Yu, Si
AU - Hu, An Bin
AU - Ma, Yi
AU - Huang, Jie Fu
PY - 2008/8
Y1 - 2008/8
N2 - Objective: To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro. Methods: Myeloid monocytes derived from 6-week-old C57BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-α (TNF-α) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 μg/ml lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD40, CD80, CD86, and MHC-II were tested by flow cytometry. IL-1β, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-S in vitro. Results: The shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 μm. The length of smDC dendrite was between 5 to 10μm. smDC, iDC, and mDC all expressed high level of CD11c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1β, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1β and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05). Conclusions: smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-α in vitro.
AB - Objective: To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro. Methods: Myeloid monocytes derived from 6-week-old C57BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-α (TNF-α) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 μg/ml lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD40, CD80, CD86, and MHC-II were tested by flow cytometry. IL-1β, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-S in vitro. Results: The shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 μm. The length of smDC dendrite was between 5 to 10μm. smDC, iDC, and mDC all expressed high level of CD11c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1β, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1β and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05). Conclusions: smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-α in vitro.
KW - Culture
KW - Identification
KW - Semimature dendritic cell
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U2 - 10.3881/j.issn.1000-503X.2008.04.014
DO - 10.3881/j.issn.1000-503X.2008.04.014
M3 - Article
C2 - 18795615
AN - SCOPUS:52649097109
VL - 30
SP - 430
EP - 435
JO - Acta Academiae Medicinae Sinicae
JF - Acta Academiae Medicinae Sinicae
SN - 1000-503X
IS - 4
ER -