We obtained leukemia macrophages (raw264.7) from Cyagen Biosciences (Wuhan, China). Rat nucleus pulposus cells were extracted from the caudal intervertebral disc of 4-week-old SD rats, as previously reported [54 (link)]. The cells were then grown in DMEM high glucose or DMEM/F12 (Cyagen, Wuhan, China) with 1% penicillin–streptomycin (P/S, Cyagen, Wuhan, China) and 10% fetal bovine serum. The culture media was then replaced every two days, while the cells were maintained at 37 °C and 5% carbon dioxide. Extracts were prepared from each group according to the following steps: 400 µL of hydrogel (height: 5 mm, diameter: 10 mm) was soaked in 3 mL of medium and cultured at 37 °C for 24 h. Extraction solution was filtered via a 0.22 µm filter membrane. The culture medium was then gathered, and fetal bovine serum and P/S were added as necessary. Prior to use, the obtained media were kept at 4 °C [55 (link)].
Dmem high glucose
Manufactured by Cyagen
DMEM high glucose is a basal medium that contains a high concentration of glucose (4.5 g/L) to support the growth and maintenance of a variety of cell types in vitro. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell culture.
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2 protocols using dmem high glucose
1
Leukemia Macrophage and Intervertebral Disc Cell Culture
2
Chondrogenic Differentiation of hUC-MSCs
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The hUC-MSCs were purchased from Cyagen Co. Ltd. (Guangzhou, China). The number of passages of cells used for the in vitro and in vivo experiments was controlled to within eight. Proliferation and differentiation medium were used. The proliferation culture medium was Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 5% fetal bovine serum (Gibco, Franklin Lakes, NJ, USA) and 1% P/S (Gibco). The medium used for hUC-MSC chondrogenic differentiation was purchased from Cyagen, with DMEM high-glucose supplemented with dexamethasone (100 nM), ascorbic acid (50 mg/mL), and sodium pyruvate (100 mg/mL). And 10 ng/mL TGF-β3 was added in fresh cell culture medium ready to use.
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