hBMSCs were purchased from Procell Life Science & Technology (CP-H166, Wuhan, China) and Cyagen Biosciences (HUXMA-01001, Guangzhou, China) and cultured in MSC medium (Cyagen, China) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin, 1% glutamine (Cyagen, China) in humidified air of 5% CO2 at 37°C. The purchased cells were accompanied by quality reports, including flow cytometry identification, which revealed that the hBMSCs were positive for CD29, CD44, CD73, and CD105, and negative for CD34, CD11b, and CD45. The purchased hBMSCs could differentiate into osteoblasts, adipocytes, and chondrocytes under specific inductive conditions. When cells reached 80-90% confluence, subculture was performed at a ratio of 1:2 or 1:3, and the medium was replaced every 2 days. After being cultured to P2-P4, the cells were plated in a 6-well plate at a density of about 1×105 cells/well. When cell confluence reached roughly 70%, hBMSCs osteogenic induction medium (Cyagen, China) containing dexamethasone, vitamin C, and β-sodium glycerophosphate was added, and was changed every 3 days.
Penicillin
Manufactured by Cyagen
Sourced in China, United States
Penicillin is a laboratory-produced antimicrobial agent used for inhibiting the growth of certain bacteria. It functions by disrupting the synthesis of the bacterial cell wall, leading to cell lysis and death.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Cyagen (5 mentions)
Glutamine, by Cyagen (3 mentions)
Msc medium, by Cyagen (1 mentions)
Fetal bovine serum (fbs), by Cyagen (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Si nc, by GenePharma (1 mentions)
Transfection reagent, by Promega (1 mentions)
Basal medium for human mesenchymal stem cells, by Cyagen (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
8 protocols using penicillin
1
Osteogenic Differentiation of hBMSCs
2
Osteogenic and Adipogenic Differentiation of MSCs
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For osteogenic differentiation, MSCs were cultured in osteogenic differentiation medium containing 10% FBS, 0.2 mmol/L ascorbate, 10 mmol/L b-glycerolphosphate, 0.1 mmol/L dexamethasone, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Cyagen, Guangzhou, China) at the confluence of 60–70%. Osteogenic medium was changed every 3 days. After induction for 21 days, cells were fixed by 4% formaldehyde, and then stained with Alizarin Red S to assess calcium deposits.
MSCs at 100% confluence were used for adipogenic differentiation. Cells were first cultured in adipogenic induction medium supplemented with 10% FBS, 1 mmol/L dexamethasone, 100 mmol/L indomethacin, 0.5 mmol/L methyisobutylxanthine, 10 mmol/L insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 3 days, followed by cultivating in maintenance medium consisting of 10% FBS, 5 mg/ml insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 24 h. After 4 cycles of induction/maintenance exchange, MSCs were cultured in maintenance medium for 6 days and finally fixed by 4% formaldehyde. Lipid droplets were examined using oil red O solution staining.
MSCs at 100% confluence were used for adipogenic differentiation. Cells were first cultured in adipogenic induction medium supplemented with 10% FBS, 1 mmol/L dexamethasone, 100 mmol/L indomethacin, 0.5 mmol/L methyisobutylxanthine, 10 mmol/L insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 3 days, followed by cultivating in maintenance medium consisting of 10% FBS, 5 mg/ml insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 24 h. After 4 cycles of induction/maintenance exchange, MSCs were cultured in maintenance medium for 6 days and finally fixed by 4% formaldehyde. Lipid droplets were examined using oil red O solution staining.
3
Adipose-Derived Cell Isolation Protocol
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Adipose tissues were drained of blood, and then digested for 45 min at 37°C by shaking in an equal volume of 0.25% Collagenase I (Sigma, USA). Digestion was terminated by adding complete culture media consisting of Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, and 0.1% penicillin (Cyagen Biosciences, Guangzhou, China). Undigested tissues and unneeded oils were filtered out using a screen mesh, and the resulting cell suspension was centrifuged for 5 min at 1200 rpm. The pellet was then immediately resuspended in fresh complete medium and recentrifuged. Finally, cells were incubated at 37°C and 5% CO2, and imaged after 2 or 3 days on an Olympus CKX41 microscope.
4
Immortalized Epithelial Cell Characterization
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All experiments between Mar 2017 and Jun 2018 were performed at Ningbo University, China in this study. The immortalized proximal tubule epithelial cell line (HK-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and Human Bone Marrow Mesenchymal Stem Cells (MSCs) were purchased from Cyagen Biosciences Inc. (SuZhou, China). HK-2 cells were cultured in DMEM (HyClone, UT, USA) medium supplemented with 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China) and MSCs were cultured in Human Bone Marrow Mesenchymal Stem Cell Basal Medium supplemented with 10% Human Bone Marrow Mesenchymal Stem Cell-Qualified Fetal Bovine Serum, 1% penicillin, 1%U/mL streptomycin and 1% Glutamine (Cyagen Biosciences, SuZhou, China). All cells were cultured in a 37 °C humidified chamber with 5% CO2 (20 (link)).
SiNC (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′) and siHGF (sense: 5′-GCACACCAAUGUGCUAAUATT-3′, anti-sense: 5′-UAUUAGCACAUUGGUCUGCUGCTT3-′) were purchased from GenePharma (Shanghai, China). Transfection Reagent (Promega Corporation, USA) were used to transfect siRNA according to the manufacturer’s instructions.
SiNC (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′) and siHGF (sense: 5′-GCACACCAAUGUGCUAAUATT-3′, anti-sense: 5′-UAUUAGCACAUUGGUCUGCUGCTT3-′) were purchased from GenePharma (Shanghai, China). Transfection Reagent (Promega Corporation, USA) were used to transfect siRNA according to the manufacturer’s instructions.
5
Rat Mesangial Cell Culture Protocol
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The rat mesangial cell line (HBYZ-1) was obtained from China Center for Type Culture Collection (China). Cells were cultured in DMEM in an incubator aerated with 95% humidified air/5% CO2 at 37°C, supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin, Cyagen Biosciences (Guangzhou) Inc., China).
6
Optimizing hBMSCs Culture and Differentiation
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The hBMSCs were purchased from Cyagen Biosciences Inc. (USA) and seeded using the basal medium for human mesenchymal stem cells (Cyagen Biosciences, USA) added with glutamine (Cyagen Biosciences, USA), 10% hBMSCs-specific FBS (Cyagen Biosciences, USA), 100 mg/ml streptomycin and 100 U/ml penicillin (Cyagen Biosciences, USA). The culture protocol was performed in an incubator following the method described in our previous work [18 (link)]. Since our previous study showed that 2.59 and 15.92 ppm optimal concentrations of Si ions in the medium, respectively, enhance the proliferation and osteogenic differentiation of hBMSCs [15 (link)], we selected the Si ions concentrations of 2.59 and 15.92 ppm for cells proliferation and osteogenic differentiation experiments, separately.
7
Isolation and Culture of Murine Astrocytes
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Murine cerebral cortical cells from a 1-day-old rat were dissociated after a 30 min trypsinization (0.15%) in D-Hanks. The cells were centrifuged, washed, and plated in 75 cm2 culture flasks (1.5 × 105/cm2) in Dulbecco's Modified Eagle Medium (DMEM)/F12 containing 12% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), and B27 (Cyagen Biosciences, Suzhou, Jiangsu, China). The medium was replenished every 2 to 3 days after plating. On day 7 of culture, floating microglial cells and oligodendrocytes were removed after shaking at 280 rpm and 37°C for 18 h. Astroglia cells were harvested after trypsinization (0.15 % trypsin in Hank’s Balanced Salt Solution, HBSS) for 30 min. After adding FBS (final concentration 12%), centrifugation, and washing, the cells were seeded into new flasks with DMEM/F12 followed by a medium change after 24 h. This subculture procedure was repeated weekly 2 - 3 times to remove residual oligodendrocytes and microglia in order to achieve a highly purified astrocyte culture. The cells were plated into 48-well cell culture plates (1 × 105 cells/well), under normal or 20 g/L D-galactose conditions.
8
Evaluating HUVEC Viability with Extract Exposure
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The ECM basal medium containing 5% fetal bovine serum (Cyagen US Inc., USA), 1% penicillin (Cyagen US Inc., USA), and 1% streptomycin and glutamine (Cyagen US Inc., USA) was used to culture the HUVECs. The HUVECs (four passages) were seeded in a 96-well plate (600 cells per well) and cultured at 37°C with 5% CO2 for 24 hours. Then, the culture medium was replaced by the medium containing extracts with different concentrations. The extract-containing medium was refreshed every 2 days. At selected time points (1, 3, and 5 days), cell viability was evaluated by CCK-8 assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Japan) according to the manufacturer’s instruction. The culture medium was replaced by mixture medium (CCK-8:medium = 1:9). After 1.5 hours, the absorbance of the reaction product was measured under 450 nm using an enzyme-linked assay microplate reader (Epoch, BioTek, Winooski, VT, USA).
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