Temporal release of IGF-1 was determined for meshes containing 0, 50, and 100 ng/mg IGF-1 (n=4 samples/group). Briefly, disks (Ø 10 mm) were cored from the mesh using a biopsy punch (Sklar) and sterilized with ultraviolet light (15 min/side). The disks were immersed (~2.2 mg/mL) in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific) with 1% ITS + premix (BD Biosciences), 50 μg/mL proline, 0.1 μM dexamethasone, 0.9 mM sodium pyruvate, and 50 μg/mL ascorbic acid (all from Sigma-Aldrich). The samples were incubated at 37 °C and 5% CO2, with media collected and replaced every 3 days. Supernatant IGF-1 concentration was quantified via enzyme-linked immunosorbent assay (ELISA, R&D Systems). Briefly, the samples were added directly to assay diluent in a prepared plate and incubated for 2 h at 4 °C. Each well was washed four times before incubation for 1 h with IGF-1 conjugate at 4 °C. The conjugate was removed, the wells were washed four times, and the substrate solution was added to each well and allowed to react in the dark. Stop solution was added after 30 min, absorbance was measured at 450 and 570 nm with a microplate reader (Tecan, Männedorf, Switzerland), and the absorbance difference was used to calculate IGF-1 concentration based on a standard curve.
Proline
Manufactured by BD
Sourced in Belgium
Proline is a high-performance laboratory equipment designed for accurate and efficient processing of biological samples. It features a robust construction, user-friendly interface, and advanced technology to ensure reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Its premix, by BD (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by BD (1 mentions)
Sodium pyruvate, by BD (1 mentions)
Ascorbic acid, by BD (1 mentions)
Microplate reader, by Tecan (1 mentions)
Transforming growth factor β1 tgf β1, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
2 protocols using proline
1
Temporal IGF-1 Release Kinetics
2
Chondrogenic Differentiation of DIAS Cells
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Cells expanded separately in monolayer from those used in clonal analysis were cultured in both environments during differentiation (hypoxia → hypoxia (HH), hypoxia → normoxia (HN), normoxia → hypoxia (NH), normoxia → normoxia (NN)). The micromass differentiation protocol was modified from the procedure described by Ahrens et al. [40] (link). This method was shown to induce DIAS cell chondrogenesis in a manner similar to the aggrecan method described previously [18] (link) in an unpublished study from our laboratory. Coated surfaces were prepared in 24-well TCP plates. A sterile 0.08% chondroitin sulfate (Sigma) solution was prepared and 20 µl was dropped into each well and allowed to dry overnight.
DIAS cells were suspended in chondrogenic medium consisting of base medium with 50 µg/ml ascorbic acid-2-phosphate (Acros Organics, Geel, Belgium), 0.4 mM proline (Acros), 50 mg/ml ITS+ Premix (BD Biosciences, Bedford, MA), 10−7 M dexamethasone (Sigma), 10 ng/ml transforming growth factor β1 (TGF-β1) (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant human insulin-like growth factor (Peprotech), and 1% FBS. 2×105 cells were seeded in a 20 µl droplet on the dried surface. After 4 hours, 500 µl of chondrogenic medium was carefully added around the condensed cell mass, and 250 µl of media was exchanged every other day for 14 days.
DIAS cells were suspended in chondrogenic medium consisting of base medium with 50 µg/ml ascorbic acid-2-phosphate (Acros Organics, Geel, Belgium), 0.4 mM proline (Acros), 50 mg/ml ITS+ Premix (BD Biosciences, Bedford, MA), 10−7 M dexamethasone (Sigma), 10 ng/ml transforming growth factor β1 (TGF-β1) (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant human insulin-like growth factor (Peprotech), and 1% FBS. 2×105 cells were seeded in a 20 µl droplet on the dried surface. After 4 hours, 500 µl of chondrogenic medium was carefully added around the condensed cell mass, and 250 µl of media was exchanged every other day for 14 days.
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