Commercial bone mesenchymal stem cells (Cyagen, MUBMX‐01001) were cultured in normoxic or hypoxic conditions, as described below. HEK293T cells (ATCC) were cultured in normoxic conditions, as described below.
Mubmx 01001
Manufactured by Cyagen
Sourced in China, United States
MUBMX-01001 is a laboratory equipment product. It serves a core function in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Mubmx 90011, by Cyagen (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Muxmx 90011, by Cyagen (2 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Facscanto 2, by BD (1 mentions)
3.0 m pore polycarbonate membrane, by Corning (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Cayman Chemical (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
D1756, by Merck Group (1 mentions)
Phr1008, by Merck Group (1 mentions)
Mc3t3 e1 cells, by Cyagen (1 mentions)
5 aza, by Merck Group (1 mentions)
Mucmx 90011, by Cyagen (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Trypsin 0.04 edta, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Guxmx 90011, by Cyagen (1 mentions)
22 protocols using mubmx 01001
1
Culturing Mesenchymal Stem Cells Under Normoxia and Hypoxia
2
Isolation and Culture of Mouse BMSCs
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Mouse BMSCs cell line (MUBMX‐01001; Cyagen Biosciences) was cultured in Mouse Mesenchymal Stem Cell Growth Medium (MUBMX‐90011; Cyagen Biosciences). Primary Mouse BMSCs were isolated from 1‐month‐old male C57/B6 mice according to the previous described method.8 , 9 Briefly, mice were anaesthetized using 5 μL/g 5% pentobarbital sodium. Then, the femurs and tibias were dissected from mice and put in the PBS with 100 U/mL penicillin and 100 µg/mL streptomycin. After washing the bone tissues in the PBS with 100 U/mL penicillin and 100 µg/mL streptomycin three times, the both ends of bones were cut off and the bone marrow were flushed out through culture medium in syringe. Then, the cells were cultured in DMEM with 5% FCS supplement with 100 U/mL penicillin and 100 µg/mL streptomycin overnight. Next day, the non‐adherent cells were removed using PBS to obtain purified BMSCs.
3
CFSE-based Proliferation Assay for Lymphocyte-MSC Co-culture
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After labelling with CFSE (Cayman Chemical Company, Ann Arbor, MI, USA), splenic lymphocytes (5 × 105 cells/well) from BALB/c mice were incubated on 24-well plates coated with an anti-mouse CD3 antibody (clone; 145-2C11) with the same number of MSCs from BALB/c mice (MUBMX-01001, Cyagen Biosciences Inc, Santa Clara, CA, U.S.) (lymp + MSC) and without MSCs (lymp) using 1000 μl of RPMI 1640 containing 10% FBS. To assess the effects of MSC-secreted soluble factors, lymphocytes and MSCs were also co-cultured without direct cell–cell contact using transwell inserts (3.0-µm pore polycarbonate membrane; Corning Inc., Armonk, NY, USA) and the MSCs were removed before performing the assay (lymp + SF). An anti-mouse CD28 antibody (clone; 37.51, 100 ng/ml) was added to each well of the CD3-coated plate. After incubation for 72 h, the cells were dissociated with 0.25% trypsin-EDTA (Thermo Fisher Scientific) and stained with anti-mouse CD3 antibodies for 30 min, and then assayed using a FACS Canto II (Becton Dickinson). The mitotic index was provided as the sum of the mitotic events in all generations divided by the calculated number of original parent cells. A list of antibodies used can be found in Table SI.
4
Osteogenic Induction with Dopamine Receptor Modulation
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The MC3T3-E1 cells (Rockville, Maryland, USA) and BMSC cells (MUBMX-01001, Cyagen, Guangzhou, China) were grown in medium containing 10% FBS (16140071, Rockville, USA). Osteogenic induction medium (DMEM, SH30022.01, Cytiva, Pittsburgh, USA) supplemented with 10% FBS, 100 nm dexamethasone (D1756, Sigma-Aldrich, MO, USA), 10 mm β-glycerophosphate (G9422, Sigma-Aldrich, MO, USA), and 50 mm vitamin C (PHR1008, Sigma-Aldrich, MO, USA). To mimic the osteolytic microenvironment, 5 μg/cm2 of titanium particles were added to the culture medium. Then, the cells were pretreated with either a D1R agonist (10 μM, SKF38393) or a D1R agonist (10 μM) plus an inhibitor (0.2 nm, SCH23390).
5
Osteogenic and Adipogenic Differentiation of Mouse BMSCs
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Mouse BMSCs (MUBMX‐01001) were obtained from Cyagen Inc., and cultured at 37°C, 5% CO2 in Alpha‐MEM medium contained 10% fetal bovine serum (Gibco), 2 mmol/l L‐glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. BMSCs were then treated with either osteogenic differentiation‐inducing medium (formulated by adding 10 mM β‐sodium glycerophosphate, 0.1 μM dexamethasone, and 50 mg/ml ascorbic acid to the growth media) or adipogenic differentiation‐inducing medium (formulated by adding 1% double antibody, 10 mM 3‐isobutyl‐1‐methylxanthine, 10 mM indomethacin, 10 nM dexamethasone to high sugar MEM media) to induce osteogenic or adipogenic differentiation respectively.23 Alizarin red S (ARS) staining and oil red O (ORO) staining were performed to detect osteogenic and adipogenic differentiation after culture with a corresponding inducing medium. BMSCs treated with growth media were served as controls. After induction, cells were harvested for further analysis. For 5‐Aza‐2′‐deoxycytidine (5‐Aza) treatment, cells were treated with 10 µM 5‐Aza (Sigma) for 96 h and the medium was replaced every 24 h.
6
Cultivation and Characterization of BMSCs
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BMSCs were purchased from Cyagen Biosciences (MUBMX-01001). Then, the cells were cultured in Mouse Mesenchymal Stem Cell Growth Medium (MUCMX-90011, Cyagen Biosciences) and cultured at 37°C and 5% CO2. CD34 and CD44 surface markers were used for BMSC analysis.
7
Isolation and Culture of Mouse Bone Marrow Stromal Cells
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Female BALB/c mice (aged 6 weeks and weighing 18–22 g) were purchased from Chengdu Dashuo Biological Co., Ltd. (Sichuan, China). The mice were placed in an isolated environment with a temperature of 22 °C–24 °C and a humidity of 40%–60% in a 12 h light-dark cycle and were provided clean drinking water and chow. In this study, all animal experiments were conducted according to the ethical standards for experimental animals (Medical Ethics Committee of Hebei Provincial Hospital of Traditional Chinese Medicine, No: 2021-KY-017-01). Mouse BMSCs were purchased from Cyagen Biosciences (China, MUBMX-01001).
8
Quantitative Alizarin Red Staining
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BMSCs were cultured with osteogenic inductive medium (MUBMX-01001, Cyagen, China). After 21 days, the mineralized nodules were stained with 2 % Alizarin Red (A5533, Sigma–Aldrich). After solubilizing in 10 % cetylpyridinium chloride (8,400,080,025, CPC, Sigma–Aldrich) for 30 min at room temperature, areas of the Alizarin Red stain were measured by the microplate reader at a wavelength of 560 nm.
9
Lentivirus-mediated miR-27b-3p and FGF2 modulation in mouse BMSCs
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Bone marrow-derived mesenchymal stem cells (BMSCs) from C57BL/6 mice were purchased from Cyagen Biosciences (MUBMX-01001, Santa Clara, CA, USA). The BMSCs were thawed and cultured in OriCell Mouse Mesenchymal Stem Cell Growth Medium (OCMM, MUXMX-90011, Cyagen Biosciences, USA) at 37℃ in 5% CO2. After infection with lentivirus carrying miR-27b-3p mimic or FGF2 overexpression plasmid, the BMSCs were passaged in OCMM at 37℃ in 5% CO2. Briefly, when the BMSCs grew to a monolayer of 80∼90% confluence, the media were aspirated. The BMSCs were rinsed with phosphate saline buffer (PBS, P5493, Sigma-Aldrich, St. Louis, MO, USA) for two or three times and then detached by 0.25% Trypsin-0.04% EDTA (T4049 Sigma-Aldrich, USA). The detached BMSCs were resuspended by OCMM and transferred to new flasks. The 6th or 7th passage of BMSCs was used for the following experiments except lentivirus infection.
293 T cells were purchased from Procell (CL-0005, Wuhan, China) and cultured in Dulbecco’s Modified Eagle Media (A4192101, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, F8687, Sigma-Aldrich, USA), 1% penicillin/streptomycin (V900929, Sigma-Aldrich, USA) and 1% Glutamine (G7513, Sigma-Aldrich, USA).
293 T cells were purchased from Procell (CL-0005, Wuhan, China) and cultured in Dulbecco’s Modified Eagle Media (A4192101, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, F8687, Sigma-Aldrich, USA), 1% penicillin/streptomycin (V900929, Sigma-Aldrich, USA) and 1% Glutamine (G7513, Sigma-Aldrich, USA).
10
Culturing Mice Bone Marrow Stromal Cells
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Mice BMSCs (MUBMX-01001, Cyagen, Guangzhou, China) planted in the wells of 24-well plates were cultivated for 24 h with mice BMSC specific medium (MUXMX-90011, Cyagen) placed in a cell incubator (51032124, Thermo Fisher Scientific, Waltham, MA, USA) under 5% CO2 and at 37°C. The cultured cells were then diluted to a 4 × 104/mL solution for further use.
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