MC3T3-E1 cells were gifted by the Institute of Oral Diseases, Nanjing Medical University, and routinely cultured in ascorbic acid (AA)-free modified α-MEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Thermo Fisher Scientific, USA). A final concentration of 25 mmol/L glucose was used to generate the HG condition. To produce this medium, 19.5 mmol/L D-glucose powder (Biofroxx, China) was added to α-MEM medium, which already contains 5.5 mmol/L D-glucose. For osteogenic differentiation, MC3T3-E1 cells were cultured in OriCell MC3T3-E1 cell osteogenic differentiation medium containing 50 μg/mL ascorbic acid, 4 mmol/L β-glycerolphosphate, and 100 nmol/L dexamethasone (Cyagen Biosciences, China) for 14 days with or without HG treatment. Fresh medium was applied every three days.
β glycerol phosphate
Manufactured by Cyagen
Sourced in China
β-glycerol phosphate is a chemical compound that functions as a source of phosphate ions. It is commonly used in cell culture media and other laboratory applications that require a phosphate buffer system.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Cyagen (4 mentions)
Ascorbic acid, by Cyagen (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
N2 dmi8, by Leica (1 mentions)
Oil red o solution, by Solarbio (1 mentions)
Alizarin red solution, by Cyagen (1 mentions)
Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Zoom stereo microscope, by Canon (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
5 protocols using β glycerol phosphate
1
MC3T3-E1 Osteogenic Differentiation Under High Glucose
2
Multilineage Differentiation of Bone Marrow Stromal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In osteogenic differentiation, BMSCs were seeded in 48-well plates as 2 × 104/well, and the medium was replaced with osteogenic induction medium (Minimum Essential Medium α +10% fetal bovine serum (FBS) + 0.1% dual antibiotics +0.1 μmol/L dexamethasone +50 μmol/L ascorbic acid +10 mmol/L β-glycerol phosphate) (Cyagen, China) at 48h. After culturing for 14 days, BMSCs were stained with alizarin red solution (Cyagen, China) and observed (Leica, N2-DMi8, Germany). In adipogenic differentiation, BMSCs were cultured with adipogenic differentiation induction medium (10% FBS +1% glutamine +0.2% insulin +0.1% IBMX +0.1% rosiglitazone +2.5% dexamethasone) (Cyagen, China). Oil red O solution (Solarbio, China) was added to stain the lipid droplet at 21 days. As for chondroblast differentiation, BMSCs were cultured with chondrogenic induction medium (DMEM medium + 0.1 μmol/L dexamethasone +1.25 mg/mL bovine serum albumin +1 mmol/L sodium pyruvate + ITS +10 ng/mL TGF-β+37.5 μg/mL vitamin C+ 1 ng/mL β-FGF) (Cyagen, China) for 14 days, and the cartilage balls were sliced and stained with cartilage staining solution (Cyagen, China).
3
Hydrogel Encapsulation of hAMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RADA-RGD/TTS/FOG solutions (1% wt/vol) were mixed at a ratio 7:3 with RADA16 solution (1% wt/vol), respectively, and sonicated for 20 min to break the molecular interaction. All the mixed peptide solutions were made at least 2 days prior to the cell encapsulation process. The hAMSCs were harvested, washed and resuspended in 10% sterile sucrose at a density of 2 × 106 cells/ml. Then the equivalent volume of the mixed peptide solution was added to the cell suspension to trigger hydrogel formation. After quickly mixing, the freshly made hydrogel was pipetted into each cell of the multi-well plate before culture medium was added. Depending on the experiments, cells were cultured for 3 − 21 days. The medium was changed regularly. For mineralization experiment and gene expression analysis, the growth medium was replaced with human umbilical cord MSC osteogenic differentiation medium (Cyagen Biosciences, 2 mmol/l β-glycerol-phosphate, 50 µmol/l ascorbic acid, 0.1 µmol/l dexamethasone). The differentiation medium was changed every 3 − 4 days.
4
Evaluating BMSC Osteogenesis by ALP Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The differentiation of BMSCs in the early stage of osteogenesis was evaluated by alkaline phosphatase (ALP) activity. First, 6 × 105 BMSCs were seeded vertically on scaffolds in 24-well plates and then cultivated in an osteoinductive medium containing 50 µg/ml ascorbic acid, 10 mM β-glycerol phosphate and 10−8M dexamethasone (Cyagen Biosciences, America) for 7 or 14 days. Then, BMSCs were lysed using 100 μL of RIPA lysis buffer. The ALP activity of the BMSCs was evaluated with an Alkaline Phosphatase Assay Kit (Beyotime, China) according to the manufacturer’s protocol. Briefly, BMSCs were incubated with the assay buffer and the samples were centrifuged to eliminate insoluble substances. Then, p-nitrophenylphosphatase liquor was mixed with the cell lysate, and the mixed solution was incubated at 37°C for 30 min. The concentration of p-nitrophenyl was determined by measuring the absorbance at 405 nm using a microplate reader (Synergy 2; BioTek, USA) and analyzing the result using a standard curve.
5
Osteogenic Differentiation Assay for BMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the evaluation of the osteogenic differentiation, the BMSCs were seeded on PEI and 3D PEI samples at a density of 1 × 104 cells/ml in 24-well culture plates with DMEM. Then, the medium was replaced by the osteogenic medium (basic culture medium containing 50 mg/L ascorbic acid, 10−8 M dexamethasone, and 10 mM β-glycerol phosphate; Cyagen, China).
After osteogenic induction for 7 days, the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) was employed to quantify the alkaline phosphatase (ALP) secretion by staining the samples according to the manufacturer's instructions. Images were acquired by a zoom stereo microscope (Canon, Japan). After induction for 14 days, the samples and cells were fixed with 4% paraformaldehyde for 20 min at 4°C. Fixed samples were washed twice with PBS for 3 min, and the Alizarin Red kit was added into the well with samples. After the samples were stained with the Alizarin Red kit for 40 min in dark, the samples were washed again with PBS, and the images were observed via a zoom stereo microscope (Canon, Japan).
After osteogenic induction for 7 days, the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) was employed to quantify the alkaline phosphatase (ALP) secretion by staining the samples according to the manufacturer's instructions. Images were acquired by a zoom stereo microscope (Canon, Japan). After induction for 14 days, the samples and cells were fixed with 4% paraformaldehyde for 20 min at 4°C. Fixed samples were washed twice with PBS for 3 min, and the Alizarin Red kit was added into the well with samples. After the samples were stained with the Alizarin Red kit for 40 min in dark, the samples were washed again with PBS, and the images were observed via a zoom stereo microscope (Canon, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!