Cre-EGFP and EGFP adenovirus and DLX5 and EGFP lentivirus were purchased from Hanbio Biotechnology Co. Ltd (Shanghai). For adenoviruses, 2 × 105 BMSCs were seeded in 12-well plates in α-MEM for 24 h, then BMSCs were infected with adenovirus expressing either Cre-EGFP recombinase or EGFP at a multiplicity of infection of 30. After 4–6 h, BMSCs were cultured in osteogenic medium (Cyagen, MUBMD-90021). For lentivirus, 2 × 105 BMSCs were seeded in 12-well plates in α-MEM for 24 h, then BMSCs were infected with lentivirus expressing either DLX5 recombinase or EGFP at a multiplicity of 20. After 24 h, BMSCs were cultured in osteogenic medium (Cyagen, MUBMD-90021).
Mubmd 90021
Manufactured by Cyagen
Sourced in United States
The MUBMD-90021 is a laboratory equipment designed for cell culture applications. It is a precision incubator that provides a controlled environment for maintaining optimal conditions for cell growth and proliferation. The core function of this product is to regulate temperature, humidity, and CO2 levels to create an ideal environment for cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Mubmd 90031, by Cyagen (3 mentions)
Mubmx 90011, by Cyagen (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
G1262, by Solarbio (1 mentions)
G1452, by Solarbio (1 mentions)
Alizarin red s, by Solarbio (1 mentions)
Ab76956, by Abcam (1 mentions)
8 protocols using mubmd 90021
1
Osteogenic Differentiation of BMSCs
2
Osteogenic Differentiation of BMSCs
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BMSCs were cultured in complete growth medium (MUBMX-90011, Cyagen Biosciences) and incubated at 37°C under 5% CO2. For osteogenic differentiation, the cells were cultured in osteogenic induction medium (MUBMD-90021; Cyagen Biosciences). The cells were maintained by the addition of fresh osteogenic induction medium every 2–3 days.
3
Adipogenic and Osteogenic Differentiation of ADSCs
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Adipogenic and osteogenic differentiation of ADSCs were performed as previously reported.7 For adipogenesis, cells were incubated in adipogenic medium (Cyagen, MUBMD‐90031) for 21 days. The medium was changed every three days. Adipogenesis was assessed by Oil Red O solution to stain lipids. For osteogenesis, cells were incubated in osteogenic medium (Cyagen, MUBMD‐90021) for 21 days. The medium was changed every three days. Osteogenesis was evaluated by alizarin red staining solution.
4
Osteogenic Differentiation of MC3T3-E1 Cells
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MC3T3-E1 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in α-Modified Eagle’s Medium (α-MEM, SH30265.01, Hyclone, USA) with 10% fetal bovine serum (FBS, 10099141, Gibco, USA) and 1% Penicillin-Streptomycin solution (15140–122, Gibco, USA). The MC3T3-E1 cells were cultured at 37 °C under 5% CO2, and during osteogenic induction, replace osteogenic differentiation medium (MUBMD-90021, Cyagen, USA). Cells were intervened by exosomes from different groups and divided into NOR group, TF group and SF group. Here, we introduce the control group (CTRL, without exosomes intervention) as a blank control.
5
Osteogenic Differentiation of VSMCs
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VSMCs (5 × 104 cells per well) were plated into 24-well plates and grew to 70% confluence in complete F12K medium, which was then changed to osteogenic medium (MUBMD-90021; Cyagen Biosciences) supplemented with solvent, YB-EVs, AB-EVs, YB-OCY-EVs, AB-OCY-EVs, OC-CM, OCYB-CM, OCAB-CM, EVs from OCYB-CM or OCAB-CM, EVs-depleted OCAB-CM, ALE (0.1−10 µM), OCALE-CM, OCAB+ALE-CM, Y-Liver-EVs, A-Liver-EVs, Y-Ser-EVs, A-Ser-EVs, or AB-EVs pre-treated with agomiR-483-5p, antagomiR-483-5p, agomiR-NC, or antagomiR-NC. The expression levels of SM22α, αSMA, RUNX2, and COL1A1 expression was assessed at 2 days after induction. ALP activity and mineralized nodule formation, respectively, were detected at 3 and 15 days after induction.
6
Osteogenic and Adipogenic Differentiation of BMSCs
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BMSCs (osteogenic induction: 1 × 105 cells per well; adipogenic induction: 2 × 105 cells per well) were seeded in 48-well plates and cultured for 24 h in complete medium, which was then replaced by osteogenic or adipogenic medium (MUBMD-90021 or MUBMD-90031; Cyagen Biosciences) supplemented with solvent, YB-EVs, AB-EVs, YB-OCY-EVs, AB-OCY-EVs, OC-CM, OCYB-CM, OCAB-CM, EVs from OCYB-CM or OCAB-CM, EVs-depleted OCAB-CM, ALE (10 µM; 129318-43-0; Aladdin, Shanghai, China), OCALE-CM, OCAB+ALE-CM, Y-Liver-EVs, A-Liver-EVs, Y-Ser-EVs, A-Ser-EVs, or AB-EVs pretreated with agomiR-483-5p, antagomiR-483-5p, agomiR-NC, or antagomiR-NC. The differentiation medium was changed every two days. For analyzing the expression of osteogenic or adipogenic genes, the cells were collected at 2 days after induction and processed for qRT-PCR. For detecting the formation of mineralized nodules or lipid droplets, the cells were stained with ARS solution (G1452; Solarbio) at 7 days after osteogenic induction or ORO solution (G1262; Solarbio) at 15 days after adipogenic induction. The percentages of ARS-positive (ARS+) and ORO+ areas were measured using Image-Pro Plus 6 software.
7
Metformin's Effects on BMSC Differentiation
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BMSCs were seeded and cultured in 48-well plates. When cells reached 100% confluence, the medium was replaced with osteogenic or adipogenic medium (Cyagen Biosciences, MUBMD-90021 or MUBMD-90031) with different concentrations of metformin (50, 250, 500 μmol·L−1) or equal volumes of PBS. BMSCs cultured in α-MEM with 10% FBS were used as a negative control. Half of the medium was replaced every other day. The cells were stained with Alizarin Red S (ARS) (Solarbio, G1452, Beijing, China) at 9 days of osteogenic differentiation or Oil Red O (ORO) (Solarbio, G1262) at 15 days of adipogenic differentiation. The percentages of ARS-positive (ARS+) and ORO+ areas were quantified with Image-Pro Plus 6 software.
8
Osteogenic Differentiation of Isolated Cells
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The isolated cells were cultured in complete medium at 5000 cells/cm2 in 6-well plates. When the cultured cells reached 80%-90% confluence, three wells of cells were cultured with basal complete medium (as the control group), and the other three wells of cells were cultured with osteogenic differentiation medium (MUBMD-90021, Cyagen, CHN) (as the osteogenic group). The medium was changed every 3 days. After 7-day culture, quantitative real-time polymerase chain reaction analysis (qRT-PCR) was performed for evaluating the expression of osteogenic genes (Runx2, Spp1, Bglap) in the cells. The primer sequences were listed in Table 3 . Meanwhile, Runx2 expression was evaluated by immunofluorescence assay using the anti-Runx2 antibody (ab76956, Abcam, USA). Additionally, Alizarin Red was used to stain the calcium nodules for assessing osteogenic differentiation of isolated cells after a 21-day culture. To conduct Alizarin Red assay, the cells were washed twice with PBS, followed by 10 min fixation in 70% ethanol and incubated in 0.5% Alizarin Red solution for 30 min, followed by PBS washing to remove the residual dye. The images of stained cells were obtained using a fluorescence microscope and a light microscope, respectively.
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