HepG2 cell lines (obtained from Dr. Pan, GIBH, CAS, Guangzhou, China) were maintained in Dulbecco's Modified Essential Medium (DMEM)-L (Gibico, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Nobimpex, Herbolzheim, Germany) at 37°C with humidified atmosphere of 5% CO2. ASGR1 -/-HepG2 cells were constructed by clustered regularly interspaced short palindromic repeats (CRIS-PR)/Cas9 [12] . To induce IR, the cells were incubated with 18 mM glucosamine (GLN) (Beyotime, Nantong, China) for 18 hours in DMEM-L without FBS [27, 28] , or 0.25 mM sodium palmitate (PA) (Sigma, St. Louis, MO, USA) in DMEM-H for 24 hours followed by incubation in DMEM-L for 2 hours [29] . Then, the cells were stimulated with 100 nM insulin (Sigma) for 20 minutes and harvested for quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses.
L dmem
Manufactured by Merck Group
Sourced in United States, China
L-DMEM is a powdered cell culture medium formulation developed by Merck Group. It is designed to support the growth and maintenance of various cell lines in in vitro cell culture applications. The formulation provides the necessary nutrients, vitamins, and other components required for cells to thrive in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (3 mentions)
H dmem, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Sodium palmitate pa, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Hascs, by Lonza (1 mentions)
Hascs, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Hepg2 cells, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
Ebm 2 bullet kit, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
T25 flask, by NEST Biotechnology (1 mentions)
Facscalibur, by BD (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Boster Biological Technology (1 mentions)
5 protocols using l dmem
1
Insulin Resistance Induction in HepG2 Cells
2
Fabrication of Minispheroid-Loaded Liver Tissues
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The present study used hASCs (Lonza, Basel, Switzerland) to establish an E-MS-printing process. Dulbecco's modified Eagle's medium low glucose (DMEM-L; Sigma-Aldrich, USA) containing 10% fetal bovine serum (FBS; Biowest, France) and 1% penicillin/streptomycin (PS; Gibco, USA) was used to culture the hASCs. HepG2 cells (Korean Cell Line Bank, South Korea) and HUVECs (Lonza, USA) were fabricating minispheroid-loaded liver tissues. Minimum essential medium (MEM) (Gibco, USA) containing 10% FBS, 1% PS, 25 mM HEPES (Sigma-Aldrich, USA), 25 mM Sodium Bicarbonate (Sigma-Aldrich, USA), and EBM™-2 Bullet Kit™ (EBM; Lonza, USA) containing 10% FBS and 1% PS were used to culture HepG2 cells and HUVECs, respectively. The cells were incubated at 37 °C with 5% CO2, and the culture medium was changed every two days.
3
Isolation and Characterization of Rat Bone Marrow Mesenchymal Stem Cells
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A 4-week-old female Wistar rat was selected for isolation of rat femur and tibia BMSCs by density gradient centrifugation. Rats were sacrificed by dislocating the cervical spine and placed in 75% alcohol for 15 min. Femurs and iliac bones were taken under aseptic conditions. We washed the medullary cavity of the femurs and iliac bones with 20 mL of serum-free LG-DMEM medium plus 1 U/mL streptomycin (BBI Life Science, Shanghai, China). L-DMEM (Sigma-Aldrich, MA, USA) added to 10% FBS (Gibco, MA, USA) was used to culture BMSCs in a T25 flask (NEST, Wuxi, China) with 3×105 cells/cm2.
The second-generation rat bone marrow mesenchymal stem cells were harvested to obtain a single-cell suspension at a concentration of 1×106/ml. We added 10 μl rat PE-CD90, FIFC-CD44, PE-CD29, and PE-CD45 anti-human antibodies, incubated at 4°C for 30 min and washed with PBS (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH=7.2). After fixing with 4% paraformaldehyde, we performed flow cytometry analysis (LSR II, BD Pharmingen, USA). All of the above antibodies were purchased from BD Pharmingen.
The second-generation rat bone marrow mesenchymal stem cells were harvested to obtain a single-cell suspension at a concentration of 1×106/ml. We added 10 μl rat PE-CD90, FIFC-CD44, PE-CD29, and PE-CD45 anti-human antibodies, incubated at 4°C for 30 min and washed with PBS (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH=7.2). After fixing with 4% paraformaldehyde, we performed flow cytometry analysis (LSR II, BD Pharmingen, USA). All of the above antibodies were purchased from BD Pharmingen.
4
Isolation and Characterization of BMMSCs
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The BMMSCs were prepared as previously described (18 (link)). Briefly, BMMSCs were obtained from the femoral shafts of the rats (from the rats with ONFH and the normal rats) after the muscles and extraosteal tissues were trimmed. Bone marrow was flushed and centrifuged on a 1.073 g/ml Percoll density gradient (Pharmacia, St. Louis, MO, USA). The BMMSCs obtained from the rats with steroid-related ONFH are hereon referred to as ONFH-BMMSCs. The cells were washed with PBS (Wuhan Boster Biological Technology, Ltd., Wuhan, China), seeded into 25-cm2 cell culture flasks, and cultivated in L-DMEM (Sigma) supplemented with 10% FBS (Sigma) and 20 mg penicillin-streptomycin/ml (Sigma) in a humidified 5% CO2 atmosphere at 37°C. The medium was changed every 3 days. When the cells became subconfluent, the cells were released from the culture substratum using trypsin/EDTA (0.25% trypsin and 0.02% EDTA) (Sigma). The cell surface molecules, CD44 and CD34, were analyzed on 3 cultures by flow cytometry (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
5
Isolation and Characterization of hSCAPs
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The apical papillae of teeth with underdeveloped roots were gently separated according to approved guidelines by the Stomatological Hospital Affiliated with Chongqing Medical University. The papilla tissues were digested with type I collagenase (Sigma, MO, USA) solution, and then maintained in Dulbecco’s Modified Eagle’s Medium with low glucose (L-DMEM, Hyclone, UT, USA) containing 10% fetal bovine serum (FBS, Hyclone, UT, USA), 1% penicillin-streptomycin at 37°C with 5% CO2, and change the medium at 3-day intervals. Three passages of hSCAPs were used in subsequent experiments.
Surface markers of hSCAPs were analyzed by flow cytometry on a BD Accuri C6 flow cytometer (BD Biosciences, CA, USA). Briefly, Cells were stained with FITC rabbit anti-CD90 (Sino-Biological, Beijing, China), anti-CD29 (Sino-Biological, Beijing, China), and anti-CD45 (Sino-Biological, Beijing, China). FlowJoTM software (Tree Star, Inc., Ashland, OR, USA) was applied to analyze the results with statistical calculations of the percentage of positive cells for visualization in histograms.
For osteogenic differentiation induction, hSCAPs were incubated in an osteogenic medium containing L-DMEM with 10% FBS, 100nM dexamethasone (Sigma, MO, USA), 10 mM β-glycerophosphate (Sigma, MO, USA), and 50 ug/ml ascorbic acid (Sigma, MO, USA).
Surface markers of hSCAPs were analyzed by flow cytometry on a BD Accuri C6 flow cytometer (BD Biosciences, CA, USA). Briefly, Cells were stained with FITC rabbit anti-CD90 (Sino-Biological, Beijing, China), anti-CD29 (Sino-Biological, Beijing, China), and anti-CD45 (Sino-Biological, Beijing, China). FlowJoTM software (Tree Star, Inc., Ashland, OR, USA) was applied to analyze the results with statistical calculations of the percentage of positive cells for visualization in histograms.
For osteogenic differentiation induction, hSCAPs were incubated in an osteogenic medium containing L-DMEM with 10% FBS, 100nM dexamethasone (Sigma, MO, USA), 10 mM β-glycerophosphate (Sigma, MO, USA), and 50 ug/ml ascorbic acid (Sigma, MO, USA).
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