C57BL/6 mice (8- to 12-week-old male, n = 18) were exterminated by CO2 inhalation and perfused with 5 ml of sterile DPBS. The entire aorta was harvested and cleaned under aseptic conditions and cut into pieces. Aorta rings were randomly divided into three groups and maintained in control, high Pi (2 mmol/L Pi), and high Pi (2 mmol/L Pi) plus DPD (25 μmol/L) conditions, respectively. The culturing medium was DMEM (D6171, Sigma) supplemented with 10% FBS (10,270-106, Gibco, Grand Island, NY, United States), antibiotic–antimycotic solution (A5955, Sigma), sodium pyruvate (S8636, Sigma), l-glutamine (G7513, Sigma), and 2.5 μg/ml amphotericin B (171,375, Millipore). The medium was changed every 2 days. After the 3rd, 5th, and 7th day, the aorta pieces were washed in DPBS, opened longitudinally and decalcified in 25 µL of 0.6 mmol/L HCl overnight. The Ca content was determined by using the QuantiChrom Ca-assay kit, as described earlier. The observer who performed aorta Ca measurements was blinded to the group assignment.
D6171
Manufactured by Merck Group
Sourced in United States
D6171 is a laboratory centrifuge. It is designed to separate components of a liquid mixture based on their density and size differences. The centrifuge spins the sample at high speeds, creating a centrifugal force that causes the denser components to settle at the bottom of the sample container.
Automatically generated - may contain errors
Lab products found in correlation
G7513, by Merck Group (5 mentions)
A5955, by Merck Group (5 mentions)
L glutamine, by Merck Group (4 mentions)
S8636, by Merck Group (3 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
D6429, by PAN Biotech (2 mentions)
P4333, by Merck Group (2 mentions)
Amphotericin b, by Merck Group (1 mentions)
Propidium iodide, by Biowest (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
F7524, by Merck Group (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Penicillin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
S5136, by Merck Group (1 mentions)
C8106, by Merck Group (1 mentions)
10 protocols using d6171
1
Aortic Calcification Quantification
2
Investigating Mode of Action of 1 on A2058 Cells
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For the investigation of the mode of action of 1 on the A2058 cells, cells were seed in six well plates (Nunclon™, Thermo Fisher) with a density of one million cells in three mL of Eagle’s medium (Dulbecco’s modified Eagles medium, D6171, Sigma) with 10% (v/v) FBS. Cells were incubated over night at 37 °C and 5% CO2 and medium was exchanged to two mL Eagle’s medium, the respective amount of compound or Triton™ X-100 (Sigma-Aldrich) as positive control, propidium iodide (Sigma) to a final concentration of 5 µg/mL and the wells were filled up to 3 mL with phosphate buffered saline (PBS, Dulbecco’s PBS, D8537, Sigma). One unstained control without propidium iodide and one negative control without compound were prepared. The reactions were incubated for 1 h at 37 °C and 5% CO2, media was removed, the cells were spilled with PBS buffer and trypsinated using 400 µL of trypsine solution (Trypsin-EDTA 10 ×, Biowest, Nuaillé, France) and redissolved in 1 mL of PBS with propidium iodide (5 µg/mL). The cell suspensions were transferred into sample tubes and analyzed using a Cytoflex flow cytometer (Beckman Coulter, Brea, Cal., US) and CytExpert.
3
NSCLC Cell Line Culture Protocol
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H460 and H1299 (non-small-cell lung carcinoma (NSCLC)) cells were purchased from ATCC and maintained in Dulbecco's Modified Eagle's Medium (DMEM) (D6171, Sigma Aldrich), supplemented with 10% foetal bovine serum (F7524, Sigma Aldrich), plus antibiotics (Penicillin, Streptomycin and Amphotericin B) (A5955, Sigma Aldrich) and L-Glutamine (G7513, Sigma Aldrich) at 37°C in a 5% CO2 atmosphere.
4
Murine Norovirus Infection Protocol
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Murine norovirus (MNV-1) CW1 strain was obtained from ATCC (VR-1937) and amplified in RAW264.7 (ATCC TIB-71) macrophages as reported (Hwang et al., 2014 ). The virus was purified by centrifugation at 30,000 × g for 3 h, and viral titer was determined by plaque assay in RAW264.7 cells. For in vitro infection, viral adsorption (MNV-1 MOI = 10) was done in plain DMEM (Sigma, D6171) containing 1% penicillin and streptomycin. The media was supplemented with FBS to reach 10% FBS 2 h after adding virus for further culture and downstream analyses.
5
Murine Norovirus Infection Protocol
6
Vascular Smooth Muscle Cell Calcification Assay
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Human aortic SMCs (HAoSMC; 354-05; Cell Applications Inc., San Diego, CA, United States) were maintained in growth medium (GM) that was prepared by supplementing Dulbecco’s Modified Eagle Medium (DMEM, D6171, Sigma) with 10% FBS (10270-106, Gibco, Grand Island, NY, United States), antibiotic antimycotic solution (A5955, Sigma), sodium pyruvate (S8636, Sigma) and L-glutamine (G7513, Sigma). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were grown to confluence and used from passages 5 to 8.
To induce calcification we cultured VSMCs in osteogenic medium (OM) that was obtained by supplementing GM with inorganic phosphate (Pi) (NaH2PO4-Na2HPO4, 1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L). A stock solution of Yoda1 (50 mmol/L) was prepared in DMSO. In calcification experiments Yoda1 was used at a concentration of 10 μmol/L and DMSO was used as a vehicle control. Yoda1 and Dooku1 were co-incubated for 5 min before changing the media every 2 days (Villa-Bellosta and Egido, 2017 (link)).
To induce calcification we cultured VSMCs in osteogenic medium (OM) that was obtained by supplementing GM with inorganic phosphate (Pi) (NaH2PO4-Na2HPO4, 1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L). A stock solution of Yoda1 (50 mmol/L) was prepared in DMSO. In calcification experiments Yoda1 was used at a concentration of 10 μmol/L and DMSO was used as a vehicle control. Yoda1 and Dooku1 were co-incubated for 5 min before changing the media every 2 days (Villa-Bellosta and Egido, 2017 (link)).
7
Human VIC Maintenance and Passaging
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Human VICs were obtained from Innoprot (Derio, Spain). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, D6171, Sigma, St. Louis, MO, USA) supplemented with 10% FBS (10270-106, Gibco, Grand Island, NY, USA), antibiotic antimycotic solution (A5955, Sigma, St. Louis, MO, USA), sodium pyruvate (S8636, Sigma, St. Louis, MO, USA), and L-glutamine (G7513, Sigma, St. Louis, MO, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown to confluence and used from passages 5 to 8.
8
Small RNA-seq and RNA-seq of HepG2 and HEK-293T Cells
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Human HepG2 cell line was grown in Dulbecco's modified Eagle's medium—high glucose (Sigma-Aldrich®, D6429) supplemented with 10% fetal bovine serum (PAN Biotech, 8500-P131704), 1% penicillin–streptomicin (v/v) (Sigma Aldrich®, P4333) and 1% l -glutamine (v/v) (Sigma Aldrich®, G7513). Human HEK-293T cells were grown in Dulbecco's modified Eagle's high glucose medium with HEPES (Sigma-Aldrich®, D6171) supplemented with 10% fetal bovine serum, 1% penicillin–streptomicin and 1% l -glutamine. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. To perform small RNA-seq and RNA-seq, HepG2 were seeded at 1.5 × 106 cells in six-well plates the day of siRNA transfection while HEK-293T were seeded at 6 × 105 cells in six-well plates.
To perform RNA Immunoprecipitation, HepG2 were seeded at 8 × 106 cells in 100 mM culture dishes the day of siRNA transfection.
To perform RNA Immunoprecipitation, HepG2 were seeded at 8 × 106 cells in 100 mM culture dishes the day of siRNA transfection.
9
Cell Culture Protocols for HepG2 and HEK-293T
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Human HepG2 cell line was grown in Dulbecco's Modified Eagle's medium -high glucose (Sigma-Aldrich®, D6429) supplemented with 10% fetal bovine serum (PAN Biotech, 8500-P131704), 1% penicillin-streptomicin (v/v) (Sigma Aldrich®, P4333) and 1% L-glutamine (v/v) (Sigma Aldrich®, G7513). Human HEK-293T cells were grown in Dulbecco's Modified Eagle's high glucose medium with HEPES (Sigma-Aldrich®, D6171) supplemented with 10% fetal bovine serum, 1% penicillinstreptomicin and 1% L-glutamine. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 . HepG2 were seeded at 1.5 × 10 6 cells in 6-well plates the day of siRNA transfection or at 7.5 × 10 5 cells in 6-well plates the day prior to plasmid transfection. HEK-293T were seeded at 6 × 10 5 cells in 6-well plates the day of siRNA transfection.
10
Culture of Human Aortic VSMCs
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Human aorta VSMCs (354-05; Cell Applications Inc, San Diego, CA) were maintained in DMEM (D6171; Sigma) supplemented with 10% fetal bovine serum (F2442; Sigma), antibiotic-antimycotic solution (A5955; Sigma), and l-glutamine (G7513; Sigma). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Cells were grown to confluence and used from passages 5 to 8. Experiments were performed on 2 cell lines derived from different donors. In some experiments, we cultured VSMCs in osteogenic medium that was obtained by supplementing the growth medium with inorganic phosphate (NaH 2 PO 4 -Na 2 HPO 4 , pH 7.4, 2.5 mmol/L; S5011 and S5136; Sigma) and CaCl 2 (0.6 mmol/L; C8106; Sigma).
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