At the time of confluence, primary suture derived cells, BMSCs, C2C12s, and MC3T3-E1s were seeded at a density of 4,000 cells per 0.32 cm2 well for alkaline phosphatase (ALP) assays (Sigma-Aldrich, USA). Comparison was made between control and thyroxine treated cells by kinetics of absorbance read at 405 nm on a Gen5 plate reader (BioTek, Winooski, VT, USA). ALP assay was repeated for MC3T3-E1 pre-osteoblasts with media as above supplemented with 0, 250, or 1000ng/ml of rhSFRP4 (R&D Systems, Minneapolis, MN). All assays were conducted after 7 days in culture.
Rhsfrp4
Manufactured by R&D Systems
RhSFRP4 is a recombinant human secreted frizzled-related protein 4 (sFRP4). sFRP4 is a member of the secreted frizzled-related protein family, which acts as a modulator of Wnt signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using rhsfrp4
1
Alkaline Phosphatase Activity in Cell Lines
2
Differentiation of Mesenchymal Stem Cells
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Trypsinized WJ- and BM-MSCs from P2 were induced to differentiate into adipocytes and osteocytes as previously described [6 (link), 10 (link), 14 (link)]. Adipogenesis was assessed by Oil Red O and osteogenesis by Alizarin Red and Von Kossa staining [6 (link), 14 (link)]. Furthermore, total RNA from BM- and WJ-MSCs following differentiation at P2 toward adipocytes and osteocytes was isolated, reverse transcribed and subsequently amplified by real-time RT-PCR, for the quantification of genes related to adipogenesis, namely peroxisome proliferator activated receptor-γ (PPARG) and CCAAT/enhancer-binding protein alpha (CEBPA), and osteogenesis, namely alkaline phosphate (ALP), osteocalcin (OSC), distal-less homeobox protein 5 (DLX5) and runt-related transcription factor 2 (RUNX2). GAPDH was used as internal control gene. The primer sequences and real-time RT-PCR detailed conditions have been described previously [12 (link)].
In a set of experiments, adipogenesis- or osteogenesis-related gene expression in WJ-MSCs was evaluated by real-time RT-PCR following differentiation of P2 cells in the presence or absence of 20 nM recombinant human (rh)-secreted frizzled related protein 4 (rh-sFRP4, R&D Systems) or 50 ng/ml rh-WNT1-inducible-signaling pathway protein 1 (rh-WISP1, R&D Systems), respectively.
In a set of experiments, adipogenesis- or osteogenesis-related gene expression in WJ-MSCs was evaluated by real-time RT-PCR following differentiation of P2 cells in the presence or absence of 20 nM recombinant human (rh)-secreted frizzled related protein 4 (rh-sFRP4, R&D Systems) or 50 ng/ml rh-WNT1-inducible-signaling pathway protein 1 (rh-WISP1, R&D Systems), respectively.
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