Osteoblasts were seeded in 6-well plates and incubated to 75% confluence. After 24 h following passaging, the maintenance medium was replaced with osteogenic induction differentiation medium (Cyagen, California, USA). The medium was replaced every 3 days. The cells were fixed with 10% formaldehyde for 30 min and stained with 0.1% alizarin red for 1 h at 37℃ to stain calcified nodules after differentiation.
Osteogenic differentiation induction medium
Manufactured by Cyagen
Sourced in United States, China
Osteogenic differentiation induction medium is a cell culture medium designed to induce the differentiation of cells towards the osteogenic lineage. It provides the necessary components to support the development of osteoblasts from progenitor cells.
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Lab products found in correlation
Oil red o, by Beyotime (1 mentions)
Adipogenic differential medium a, by Cyagen (1 mentions)
Paraformaldehyde (pfa), by Cyagen (1 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Alizarin red, by Cyagen (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
7 protocols using osteogenic differentiation induction medium
1
Osteogenic Differentiation of Osteoblasts
2
Osteogenic Differentiation of Cells
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Cells were seeded at a density of 2 × 104 cells/cm2 in a 6-well plate coated with 0.1% gelatin, and 2 mL of medium was added to each well, which was then cultured in a 37.5 ˚C, 5% CO2 incubator. When the degree of cell fusion reached 60%–70%, the medium in the 6-well plate was replaced with complete osteogenic induction differentiation medium (Cyagen Biosciences, Inc.), which was replaced with fresh osteogenic induction differentiation medium every three days. After 2–4 wk of induction, the cells were stained with Alizarin Red (Shanghai Beyotime Co., Ltd), and the cell mor-phology was observed under an inverted fluorescence microscope.
3
Osteogenic and Adipogenic Differentiation of CSPCs
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CSPCs were seeded onto 24-well plates at a concentration of 2 × 10^4 per well. For osteogenic differentiation, when the cells reached 80%~90% confluence, the culture media was replaced by 0.5 mL osteogenic differentiation induction medium (Cyagen Biosciences Inc., USA) containing 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.1 mM L-ascorbic acid-2-phosphate. Induction medium was replaced each other day. After four weeks, the cells were rinsed and fixed by 4% formaldehyde. Alizarin red staining was performed following the instruction of the manufacturer.
For adipogenic differentiation, medium was changed into adipogenic differential medium A (purchased from Cyagen Biosciences Inc., USA) for induction when the cultures reached full confluence. Three days later, adipogenic differential media B (Cyagen Biosciences Inc., USA) was used for 24-hour maintaining. Four cycles later, cells were incubated with adipogenic differential media B for 6 more days. Oil Red O (Beyotime, China) staining was used for lipid detecting, which would show lipid droplets in reddish-brown.
For adipogenic differentiation, medium was changed into adipogenic differential medium A (purchased from Cyagen Biosciences Inc., USA) for induction when the cultures reached full confluence. Three days later, adipogenic differential media B (Cyagen Biosciences Inc., USA) was used for 24-hour maintaining. Four cycles later, cells were incubated with adipogenic differential media B for 6 more days. Oil Red O (Beyotime, China) staining was used for lipid detecting, which would show lipid droplets in reddish-brown.
4
Osteoblastogenesis Induction and Alizarin Red Staining
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Osteoblastogenesis induction and alizarin red staining were performed as described previously [65 (link)]. BMSCs with a density of 5 × 104 cells/cm2 were seeded on the surfaces of sterilized Ti, TNrs, and Fe3O4-TNrs or into the wells without any sheets, treated with 1 mT 15 Hz SEMF for 1 h per day. BMSCs were cultured with the osteogenic differentiation induction medium (Cyagen Biosciences, CA, USA). After 21 days of culture, the cells were fixed with 4% paraformaldehyde for 20 min, and stained with alizarin red (Cyagen Biosciences, CA, USA) for 5 min, washed with PBS three times, then the cells could be placed under the microscope (EVOS FL Auto, Life Technologies, USA) to observe and photograph.
5
Adipose-Derived Stem Cell Isolation and Differentiation
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Human subcutaneous adipose tissues were washed with sterile PBS containing 1 g/L penicillin‒streptomycin (4 °C), wiped of the fascia and blood vessels, cut into small pieces, and then digested with 20 g/L collagenase type I. Then, the mixture was filtered by 100 μm cell screening, centrifuged at 300×g for 5 min and resuspended in culture medium. 3rd- to 5th-generation ADSC were incubated with antibodies (CD 73 CD 44, CD 29, CD 90, CD 34, and CD 45) and detected with flow cytometry. For osteogenesis experiments, ADSCs were seeded into six-well cell culture plate followed by osteogenic induction of differentiation at approximately 60–70% confluence. After treatment with osteogenic differentiation induction medium (Cyagen Bioscience, Inc.,China) for 3 weeks, the induced ADSC were fixed in 40 g/L paraformaldehyde and stained with Alizarin Red to view the induction results. For lipogenic differentiation, ADSC were seeded into six-well cell culture plate followed by lipogenic induction of differentiation at approximately 90–100% confluence. After treatment with lipogenic differentiation induction medium (Cyagen Bioscience, Inc., China) for 2 weeks, the ADSC were fixed in 4% PFM for more than 20 min then stained with Oil Red O to view the induction results.
6
FGFR2 Knockdown and Overexpression in MC3T3-E1 Cells
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Pre-osteoblastic murine cell line MC3T3-E1 cells were cultured under aseptic conditions using high-glucose Dulbecco’s modified Eagle medium (DMEM, Hyclone, Logan, GA, USA), containing 10% fetal bovine serum (FBS, Biological Industries, Israel) and 50 U/mL penicillin and 50 μg/mL streptomycin (Hyclone, Logan, GA, USA) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded at 6~8 × 104 cells/well in 12-well plates.
To knock down FGFR2, small interfering RNA (siRNA) specifically targeting FGFR2 were designed, (sense, GGAGUUUAAGCAGGAGCAUTT, antisense, AUGCUCCUGCUUAAACUCCTT) and siNC was used as a negative control (Shanghai Lingke Tech, Shanghai, China), transfected into MC3T3-E1 cells using JetPRIME transfection reagent (Ployplus-transfection, Illkirch, France), and incubated for 48 h. At 48 h post-transfection, the medium was replaced by osteogenic differentiation induction medium (Cyagen, Suzhou, Jiangsu, China).
As shown inSupplementary Figure S2 , wild-type (WT) FGFR2, Cys342Arg mutant (MT) FGFR2 and domain negative (DN) FGFR2 overexpression plasmids with Flag tag on the side of C-terminal were constructed by Tsingke Biotechnology Co, Ltd, China. Additionally, they were extracted by EndoFree Plasmid Midi Kit (Jiangsu Cowin Biotech Co, Jiangsu, China) and then transfected into MC3T3-E1 cells using JetPRIME transfection reagent and incubated for 24 h.
To knock down FGFR2, small interfering RNA (siRNA) specifically targeting FGFR2 were designed, (sense, GGAGUUUAAGCAGGAGCAUTT, antisense, AUGCUCCUGCUUAAACUCCTT) and siNC was used as a negative control (Shanghai Lingke Tech, Shanghai, China), transfected into MC3T3-E1 cells using JetPRIME transfection reagent (Ployplus-transfection, Illkirch, France), and incubated for 48 h. At 48 h post-transfection, the medium was replaced by osteogenic differentiation induction medium (Cyagen, Suzhou, Jiangsu, China).
As shown in
7
Osteogenic Differentiation of BMSCs
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BMSCs were seeded into a 24-well plate at 5 × 104 cells per well. When they reached 60-70% confluence, α-MEM complete medium was discarded, the cell layer was washed twice with phosphate-buffered saline (PBS; Solarbio) and osteogenic differentiation induction medium (Cyagen, Guangzhou, China) was added. The culture medium was replaced every three days. After 7 days of osteogenic induction, the osteogenic differentiation induction medium was discarded, and the cell layer was washed twice with PBS followed by fixation using 4% neutral-buffered formalin. ALP activity was measured using a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride (BCIP/NBT) alkaline phosphatase color development kit (Solarbio), according to the manufacturer's protocol. The staining results were observed under a light microscope (Olympus IX 70, Tokyo, Japan), and the area of staining was evaluated using Image J software (NIH, Bethesda, MD, USA). After 14 days of osteogenic induction, alizarin red staining was performed to evaluate calcium deposits according to the manufacturer's protocol (Cyagen). Then the staining results were observed under a light microscope (Olympus IX 70). To quantify the mineralization, alizarin red was eluted from the monolayer with 10% cetylpyridinium chloride (CPC; Sigma), and the absorbance was measured at 570 nm using a microplate reader (BioTek).
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