HepG2 and HEK293T cells were obtained from the “Vertebrate Cell Culture Collection” of the shared research facility at the Institute of Cytology, RAS. Cells were routinely cultured in DMEM High Glucose (Servicebio, Wuhan, China) containing 10% FBS (Sigma-Aldrich, São Paulo, Brazil), 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.25 μg/mL amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 incubator (NuAire, Inc., Plymouth, MN, USA). Cells were passaged when cell fusion was over 80%, about twice a week.
Dmem high glucose
Manufactured by Wuhan Servicebio Technology
Sourced in China
DMEM High Glucose is a cell culture medium that provides high levels of glucose to support the growth and maintenance of various cell types. It is a widely used formulation in the field of cell biology and biotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
Co2 incubator, by NuAire (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Corning (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Biosharp (1 mentions)
5 protocols using dmem high glucose
1
Cell Culture of HepG2 and HEK293T
2
Culturing Cell Lines and Infecting with JEV
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BHK-21 cells (hamster kidney cell line), HeLa cells (human cervical cancer cell line), BV2 cells, HEK-293T cells (human microglia cell line) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin sulfate in a 37 °C, 5% CO2 incubator. SH-SY5Y cells (human neuroblastoma cell line) were cultured in Dulbecco's modified Eagle's medium (DMEM/High Glucose) and F12 (Servicebio) (1:1) medium with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate in a 37 °C, 5% CO2 incubator.
The JEV P3 strain was stored in our laboratory. Cells were infected with JEV at an MOI of 1 for 1 h, followed by removal of the supernatant and addition of maintenance medium containing 3% FBS. For the use of inhibitors, Lip-1 was added into maintenance medium at a concentration of 20 μmol/L, and maintained throughout the experiment.
The JEV P3 strain was stored in our laboratory. Cells were infected with JEV at an MOI of 1 for 1 h, followed by removal of the supernatant and addition of maintenance medium containing 3% FBS. For the use of inhibitors, Lip-1 was added into maintenance medium at a concentration of 20 μmol/L, and maintained throughout the experiment.
3
HepG2 cell culture protocol
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HepG2 cells were obtained from the shared research facility “Vertebrate cell culture collection”, Institute of cytology RAS. Cells were routinely cultured in DMEM high-glucose (Servicebio, Wuhan, China) containing 10% FBS (Sigma-Aldrich, São Paulo, Brazil), 100 μg/mL streptomycin, 100 U/mL penicillin and 0.25 μg/mL amphotericin B (Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C in a 5% CO2 incubator (NuAire, Inc., Plymouth, MN, USA). Cells were passaged when cell fusion was over 80%, about twice a week.
4
HepG2 cell culture protocol
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HepG2 cells were obtained from the shared research facility “Vertebrate cell culture collection” Institute of Cytology RAS. Cells were routinely cultured in high-glucose DMEM (Servicebio, Wuhan, China) containing 10% FBS (Sigma-Aldrich, São Paulo, Brazil), 100 μg/mL streptomycin, 100 U/mL penicillin and 0.25 μg/mL amphotericin B (Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C in a 5% CO2 incubator (NuAire, Inc., Plymouth, MN, USA). Cells were passaged when cell fusion was over 80%, about twice a week.
5
LPS-Induced Macrophage Activation Model
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The murine RAW264.7 macrophage line was purchased from the Cell Culture Center, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in high-glucose DMEM (Servicebio) with 10% foetal bovine serum (Zeta Life, Menlo Park, CA, USA) and 1% penicillin‒streptomycin (Biosharp, Anhui, China) at 37℃ under 5% CO2. RAW264.7 cells (1×106) were treated with 1 μg/ml lipopolysaccharide (LPS, Corning, Wilmington, NC, USA) to mimic the septic environment in vitro (6, 12, and 24 h). For cell transfection, RAW264.7 cells were seeded on 60 mm-cell dishes and cultured with serum-free Opti-MEM (Gibco, Brooklyn, NY, USA) 1 h before transfection when cell confluence reached 70-80%. Then, the cells were transfected with ADAR1-overexpression adenovirus (GenePharma), ADRA1-siRNA (RiboBio, Guangzhou, China), A20-siRNA (RiboBio), miR-21 mimic, miR-21 inhibitor (RiboBio) and their negative controls through Zeta transfection reagents (Zeta Life). Further LPS treatment was performed 24 h after transfection. The cells were collected for further experiments 48 h after transfection.
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