The multilineage differentiation potential of hPVCs cultured in NG and HG conditions was evaluated at passages 3 and 4 as previously described [13 (link)]. Briefly, for adipogenic differentiation, hPVCs (4 × 104 cells/well) were plated in 12-well tissue culture plates and cultured in adipogensis differentiation medium (A10070-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. After 21 days, the cultures were stained with Oil Red O (CM-005; LifeLine Cell Technology, CA, USA) to visualize the intracellular accumulation of lipid vacuoles. For osteogenic differentiation, hPVCs (4 × 104 cells/well) were seeded in 12-well tissue culture plates and cultured in osteogenesis differentiation medium (A100-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. On day 21, the cells were stained with Alizarin Red S (Lifeline Cell Technology) to visualize the mineralization in osteogenic cell cultures.
Alizarin red s
Manufactured by Lifeline Cell Technology
Sourced in United States
Alizarin Red S is a chemical compound used as a staining agent in various laboratory applications. It is commonly used to detect and visualize the presence of calcium deposits in biological samples. The dye binds to calcium ions, producing a bright red color that can be observed under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Stempro, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Lifeline Cell Technology (2 mentions)
Mesencult basal medium, by STEMCELL (2 mentions)
Mouse mesencult adipogenic stimulatory supplement, by STEMCELL (2 mentions)
Oil red o solution, by Lifeline Cell Technology (2 mentions)
Mesencult basal medium mouse mesencult osteogenic stimulatory kit, by STEMCELL (2 mentions)
A10070 01, by Thermo Fisher Scientific (1 mentions)
5 protocols using alizarin red s
1
Multilineage Differentiation of hPVCs
2
Adipogenic and Osteogenic Potentials of HUCPVCs
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The in vitro adipogenic and osteogenic differentiation potentials of HUCPVCs were assesed as previously described (An et al., 2015 ). Briefly, HUCPVCs were plated at 4.5 × 103 cells per cm2 in 12-well tissue culture plates and cultured in the presence of adipogenic and osteogenic supplements (Gibco). The medium was changed every 3 days for 21 days. The lipid vacuoles and mineralization, respectively, were visualized by Oil Red O or Alizarin Red S (Lifeline Cell Technology) and assessed quantitatively using a spectrophotometer (BioTek Instruments).
3
Adipogenic and Osteogenic Differentiation of MSC Subsets
4
Adipogenic and Osteogenic Differentiation of MSC Subsets
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FACS sorted MSC subsets were first grown for 2 weeks in phenol red-free α-MEM plus 10% fetal bovine serum and 1% penicillin/streptomycin for 2 weeks with one half medium change every 3 days to reach 70% confluency. For adipocyte differentiation, MSC subsets were cultured in adipogenesis medium (MesenCult basal medium + mouse MesenCult adipogenic stimulatory supplement; Cat no. 05503; Stem Cell Technologies) for further 3 weeks by following manufacturer’s instructions. Briefly, medium was changed every 3–4 days and wells were then stained with Oil Red O solution (Cat no. LL-0052; Lifeline Cell Technology, Frederick, MD). For osteoblast differentiation, MSC subsets were cultured in osteogenic medium (MesenCult basal medium + mouse MesenCult osteogenic stimulatory kit; Cat no. 05504; Stem Cell Technologies) for further 3 weeks by following manufacturer’s instructions. Briefly, medium was changed every 3–4 days and wells were then stained with 2% Alizarin Red S (Cat no. CM-0058; Lifeline Cell Technology, Frederick, MD).
5
Multilineage Differentiation of PVCs
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The multilineage differentiation potentials of the PVCs were evaluated as previously described [13 ]. Briefly, PVCs from passage 3 were seeded at 4×104 cells/well in expansion medium. When the cells reached 80% confluency, they were treated with osteogenic or adipogenic induction medium (StemPro®, ThermoFisher Scientific, San Jose, CA, USA) for 21 days at which time the medium was changed every 3 days. PVC cultures were stained with Alizarin Red S (CM-0058; Lifeline Cell Technology, Carlsbad, CA, USA) or Oil Red O (CM-0055; Lifeline Cell Technology) and the dye contents were quantified using a spectrophotometer.
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