Animal use in this study was approved by Jilin University Animal Care and Use Committee. The animal experiments were carried out in accordance with requirements of the m Experimental Animal Ethics and Welfare guidelines (Permit Number: 201611). Rat bone marrow stromal cells (BMSCs) were isolated from femurs of 4-week-old rats. Briefly, bone marrow cells were flushed out using a 20 mL syringe and plated in a 25 mL cell culture flask (NEST, Wuxi, China). The cells were cultured in low-glucose DMEM (L-DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 mg/mL of streptomycin (Thermo Fisher Scientific), and 100 U/mL of penicillin (Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator. The medium was changed after 4 days of isolation of cells, and then the culture medium was changed every 3 days. The third passage of BMSCs was prepared for subsequent tests.
Cell culture flask
Manufactured by NEST Biotechnology
Sourced in China
Cell culture flasks are sterile, transparent containers used for in vitro cultivation of cells. They provide a controlled environment for cell growth and proliferation, enabling researchers to study cellular processes and maintain cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L dmem, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Protein loading buffer, by Beyotime (1 mentions)
Terminal deoxynucleotidyl transferase mediated dutp nick end labeling tunel kit, by Roche (1 mentions)
Rna keeper, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Trypsin, by Solarbio (1 mentions)
Reverse transcription kit, by Vazyme (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Hoechst staining solution, by Beyotime (1 mentions)
Sybr green reagent, by Vazyme (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
7 protocols using cell culture flask
1
Isolation and Culture of Rat BMSCs
2
In Vitro Osteoblast Cytotoxicity Assay
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Osteoblasts (MC3T3-E1) were selected for the in vitro research. The MC3T3-E1 cells were maintained in a cell culture flask (Nest) containing Dulbecco's Modified Eagle Medium (DMEM, high glucose, Gibco) with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C under 5% CO2. The culture medium was changed with fresh medium every 48 h, and the cells were digested with 0.25% trypsin-EDTA solution (Gibco).
The samples were sterilized by exposing them to UV radiation for 30 min and then incubated in culture medium for 24 h. The ratio of the extraction medium's volume to the sample's surface area was 1.25 mL cm−2. The extracted solution was used as the culture medium for the subsequent experiments. The cells with a density of 5 × 104 cells/well were injected into 96-well plates for cell viability detection. After incubation for 24 h, the extracted solution was used to replace the culture medium. As described previously [45 ], the viability of the cells that were cultured for 1, 3, and 5 days was measured by standard MTT assay.
The samples were sterilized by exposing them to UV radiation for 30 min and then incubated in culture medium for 24 h. The ratio of the extraction medium's volume to the sample's surface area was 1.25 mL cm−2. The extracted solution was used as the culture medium for the subsequent experiments. The cells with a density of 5 × 104 cells/well were injected into 96-well plates for cell viability detection. After incubation for 24 h, the extracted solution was used to replace the culture medium. As described previously [45 ], the viability of the cells that were cultured for 1, 3, and 5 days was measured by standard MTT assay.
3
Osteoblast Cell Line Growth and Characterization
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The human osteoblast cell line HCO was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. α-MEM culture solution and fetal bovine serum (FBS) were purchased from Hyclone, USA. Trypsin, penicillin-streptomycin and phosphate buffered saline (PBS) were purchased from Solarbio, China. Cell culture flask and other consumables were purchased from NEST, USA. Serum-free Opti medium and TRIzol reagent were purchased from Invitrogen, USA. Methyl thiazolyl tetrazolium (MTT) powder was purchased from Biosharp, China. Organic reagents, such as dimethyl sulfoxide (DMSO), were purchased from Shanghai Rhawn Technology Development Co., Ltd. The reverse transcription kit, transfection reagent X-treme, RNA Keeper and SYBR Green reagent were purchased from Vazyme, China. MiR-214 mimics, miR-214 AMO and its negative control (NC) and PCR primers were designed and synthesized by Shanghai Generay Biotechnology Co., Ltd. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit was bought from Roche, Switzerland. 4% paraformaldehyde, protein lysis buffer, Hoechst staining solution, BCA protein concentration assay kit and protein loading buffer were bought from Beyotime, China.
4
Cell Culture Protocol with FBS, PBS, and Media
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Fetal bovine serum (FBS), phosphate-buffered solution (PBS), penicillin-streptomycin solution, 0.25% trypsin containing 0.02% ethylenediaminetetraacetate (EDTA), Leibovitz’s L-15 medium, and RPMI 1640 medium were purchased from Gibco, Life Technology, Shanghai, China. Propidium iodide (PI) was obtained from Invitrogen, Carlsbad, CA, USA). All of the disposable consumables, such as cell culture flasks, mesh filters (45 μm), suction tips, pipettes and tubes, used in the experiment were provided by Nest Biotechnology, Wuxi, China.
5
Comprehensive Reagents and Materials
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The following reagents were used in this study: the Von Hippel-Lindau (VHL) E3 ligand (S, R, S)-AHPC-PEG3-azide (AHPC) (Sigma Aldrich), MG132 (Sigma Aldrich), MLN4924 (S7109, Selleck Chemicals), anti-nucleolin antibody (D4C7O, Cell Signaling Technology), anti-β-Tubulin antibody (9F3, Cell Signaling Technology), anti-mouse immunoglobulin (Ig)G Alexa Fluor 488 Conjugate (4408, Cell Signaling Technology), anti-rabbit IgG Alexa Fluor 594 Conjugate antibody (8889, Cell Signaling Technology), anti-VHL antibody (sc-135657, Santa Cruz), anti-Ub antibody (sc-166553, Santa Cruz), anti-β-Actin antibody (AT0001, Engibody), anti-Flag antibody (20543-1-AP, Proteintech), anti-MYC antibody (M192-3, MBL International), anti-HA antibody (M180-3, Medical & Biological Laboratory), Cell Counting Kit-8 (CCK-8) (New Cell & Molecular Biotech), Agarose and Super GelRed (S2001, US Everbright Inc), and Cell Culture flasks (NEST Biotechnology). All DNA sequences were synthesized by Hippobio Technology (Zhejiang, China) and Shenggong Biotechnology (Shanghai, China).
6
Endothelial Cell Cultivation and Treatment
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Human umbilical vein endothelial cell and the ECM were purchased from ScienCell Research Laboratories (San Diego, CA, United States). The ECM was composed of basal medium, 1% endothelial cell growth supplement, 5% fetal bovine serum, and 1% penicillin/streptomycin solution. HUVECs were seeded in cell culture flasks (25 cm2; NEST, Shanghai, China) coated with poly-L-lysine, and cultivated in an atmosphere with 95% humidity and 5% CO2 at 37°C. Cells were sub-cultured by trypsinization (0.25% trypsin, 0.5 mM EDTA) when they had grown to 80∼90% confluence. Three to six passages cells were used. The culture medium was replaced with serum-free medium for 6 h before treatment with various concentrations of 1,8-cineole with or without LPS (2.5 μg/ml) as indicated for 12 h, and the vehicle control contained serum-free medium only.
7
Transgenic Strain Construction in BALB/c Mice
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Type I strain RHΔKu80 was used to construct the transgenic strain, which was maintained in DF-1 cells (a spontaneously immortalized chicken embryo fibroblast cell line) or Vero cells (African green monkey kidney cells) at 37 °C and 5% CO2 using cell culture flasks (NEST Biotechnology, Wuxi, China). Four-week-old female BALB/c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and kept under standard conditions in accordance with the regulations specified by the Administration of Affairs Concerning Experimental Animals.
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