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Blood, Vol. 112, Issue 13, 5074-5083, December 15, 2008
Bone marrow microenvironment controls the in vivo differentiation of murine dendritic cells into osteoclasts
Blood Wakkach et al.
112: 5074
Supplemental materials for: Wakkach et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 66.1 KB)
- Figure S1. Osteoclastogenic potential of the main DC subsets (JPG, 111 KB)
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Subsets of cDCs were purified from the CD11c+ enriched cells after labeling with with APC-CD11c, FITC-IAb, PE-CD8α (clone 53-6.7) and PECy7-CD4 (clone RM4-5). The CD4−CD8−, CD4+CD8− and CD4−CD8+ DC subsets were sorted from the CD11c+IAb+ cells on a FACS Aria (Becton-Dickinson) with high purity sorting (100%). Plasmacytoid DCs (pDCs) were obtained from the splenocytes remaining after depletion of CD3+CD19+CD49b+ cells, as described above. The remaining cells were labelled with APC-CD11c, FITC-IAb and PE-B220. The CD11chighB220– cDCs and the CD11clowB220+ pDCs were sorted on a FACS Aria (Becton-Dickinson) with high purity sorting (100%). (A) The CD4−CD8−, CD4+CD8, and CD4−CD8+ subsets of cDCs and (B) the CD11clowB220+ pDCs and the CD11chighB220−cDCs were FACS purified from the spleen of normal mice and cultured onto dentin slices in the presence of M-CSF (25 ng/ml) and RANK-L (30 ng/ml) for 8 days (cDCs) or 18 days (pDCs). The TRAP activity was analyzed and TRAP+ multinucleated cells (MNC) were counted. Performing this analysis on the dentin slices revealed the presence of resorption lacunae, reflecting a resorption activity. The results were presented as the mean ± SD of 8 equivalent wells for 3 independent experiments. All cDCs subsets are not equivalent for OCL differentiation and the CD4−CD8− subset contributes much more efficiently than others to this differentiation. OCL formation from the CD11clowB220+ pDCs was less efficient and required a longer time than from CD11chighB220− cDCs. (C) Immature DCs were generated from mouse bone marrow in the presence of GM-CSF (10 ng/ml) and IL-4 (10 ng/ml). After 6 days, immature DCs were purified from the culture using CD11c+-microbeads (Miltenyi-Biotech) on a MS column. Sorted immature CD11c+ DCs were activated 24 h in the presence of LPS (1 µg/ml), CpG (1 µM) or not activated. Activation was evaluated by flow cytometry analysis with APC-CD11c and PE-CD86 antibodies. The sorted cells were then cultured for 8 days onto dentin slices in the presence of M-CSF (25 ng/ml) and RANK-L (30 ng/ml). The TRAP activity was analyzed and TRAP+ multinucleated cells (MNC) were counted. Presence of resorption lacunae was also detected. The results were presented as the mean ± SD of 8 equivalent wells. Data are representative of 2 independent experiments. Immature and matured DCs differentiated into functional OCLs, with the same efficiency indicating that maturation did not affect the osteoclastogenesis process from DCs.
- Figure S2. the expression of RANK-L and M-CSF is increased in the bone marrow of oc/oc mice (JPG, 22.2 KB)
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The expression of RANK-L and M-CSF was analyzed by real-time RT-PCR in bone marrow cells isolated from 18-day-old normal (+/+) and oc/oc mice. The results were presented as the mean ± SD of 3 mice in each group and are representative of 2 independent experiments.
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