Mouse dental papilla cells (mDPCs) were isolated from the first molars of E15.5 CD1 mice and digested for 1 h at 37 °C in a solution of 3 mg/mL collagenase type I (Worthington Biochemical, USA) and 4 mg/mL Dispase II (Roche, Mannheim, Germany). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 11995; Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; 10099141; Gibco) plus 1% penicillin and streptomycin (control medium, CM). To induce odontoblastic differentiation, mDPCs were seeded at a density of approximately 100,000 cells per well in 12-well plates and treated with odontoblastic medium (OM; CM supplemented with 10−7 M dexamethasone (Sigma–Aldrich, St Louis, MO, USA), 50 µg/mL ascorbic acid (Sigma–Aldrich) and 10 mM sodium β-glycerophosphate (Sigma–Aldrich)). Primary mDPCs at passages 2–4 were used for cell experiments. HEK293E cells were cultured in DMEM (Gibco) supplemented with 10% FBS. MC3T3-E1 cells were cultured in minimum essential medium (MEM Alpha basic; 12571; Gibco) supplemented with 10% FBS. Cells were cultured at 37 °C in a humidified atmosphere of air containing 5% CO2.
Mem alpha basic
Manufactured by Thermo Fisher Scientific
Sourced in United States
MEM Alpha basic is a powdered culture medium used for the cultivation and maintenance of mammalian cell lines. It provides essential nutrients and supports the growth of a variety of cell types. The product is designed for use in cell culture applications.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Worthington (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Dispase 2, by Roche (1 mentions)
Sodium β glycerophosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Ab19027, by Abcam (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Ab52750, by Abcam (1 mentions)
Recombinant rankl, by Thermo Fisher Scientific (1 mentions)
Actin tracker green, by Beyotime (1 mentions)
Bs c 1, by American Type Culture Collection (1 mentions)
4 protocols using mem alpha basic
1
Isolation and Differentiation of Mouse Dental Papilla Cells
2
Isolation and Characterization of Rat Bone Marrow Stromal Cells
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BMSCs were extracted
from 2 week old male
Sprague–Dawley rats.40 (link) Briefly,
the femur and tibia of euthanized rats were surgically removed, and
the bone marrow cavity was washed with basic medium to obtain BMSCs.
Flow cytometry was used to identify the cell type (Figure S1 ). An MEM-Alpha basic (Gibco, Grand Island, NY, USA)
containing 10% fetal bovine serum (Gibco) and 1% antibiotic solution
(penicillin and streptomycin) was used for culture. Cells obtained
after passages 3–5 were used for subsequent experiments. Animal
experiments were approved by the Sichuan Experimental Animal Management
Committee (Sichuan, China).
from 2 week old male
Sprague–Dawley rats.40 (link) Briefly,
the femur and tibia of euthanized rats were surgically removed, and
the bone marrow cavity was washed with basic medium to obtain BMSCs.
Flow cytometry was used to identify the cell type (
containing 10% fetal bovine serum (Gibco) and 1% antibiotic solution
(penicillin and streptomycin) was used for culture. Cells obtained
after passages 3–5 were used for subsequent experiments. Animal
experiments were approved by the Sichuan Experimental Animal Management
Committee (Sichuan, China).
3
Osteoclastogenesis Inhibition by Flavonoid Compounds
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Recombinant RANKL and M-CSF were purchased from PeproTech (Princeton, NJ, United States). Icariside I (ICS; C27H30O11; MW: 530.53), baohuoside Ⅰ (BS; C27H30O10; MW: 512.52), icariin (ICA; C33H40O15; MW: 676.68), icaritin (ICT; C21H20O6; MW: 368.38), and a Cell Counting Kit-8 (CCK-8) were purchased from Target Molecule Corp. (Boston, MA, United States). Trypsin-EDTA (0.05%), PBS, FBS, and MEM-Alpha basic were purchased from Gibco. Antibodies against p38 (#8690), p-p38 (#4511), ERK (#9102), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p65 (#8242), p-p65 (#3033), IκBα (#4812), p-IκBα (#2859), and RANK (#4845) were purchased from Cell Signaling Technology (Boston, MA, United States). Antibodies against TRAP (ab52750) and cathepsin K (ab19027) were purchased from Abcam (Cambridge, MA, United States). Antibodies against NFATc1 (66963-1-Ig), uPAR (10286-1-AP), and β-actin (66009-1-Ig) were purchased from Proteintech Group Inc. (Wuhan, China). DAPI and Actin-Tracker Green were obtained from Beyotime (Shanghai, China). Cy3-labelled goat anti-rabbit antibody was purchased from Boster (Wuhan, China). Unless noted otherwise, other reagents were of the highest purity available and were obtained from Sigma-Aldrich (St. Louis, MO, United States).
4
Cultivation and Maintenance of Fetal Lung and Kidney Cells for HAV Research
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Human fetal lung diploid fibroblastic cells (2BS, ATCC bio-53536), and African green monkey kidney cells (BS–C-1, ATCC CCL-26) were purchased from ATCC. 2BS cells were maintained in MEM Alpha basic (Gibco, USA) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS. BS-C-1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS. The two cells were grown at 37 °C in 5% CO2.
HAV vaccine strain H2 (GenBank accession no. EF406359) was kindly provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). Virus stocks were prepared in 2BS cells and stored in aliquots at −80 °C. Virus titers were determined by standard RT-qPCR as described below. HAV strain HM175/18f (GenBank accession no. M59808) was kindly provided by Prof. Qiang Ding (Tsinghua University, Beijing).
HAV vaccine strain H2 (GenBank accession no. EF406359) was kindly provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences (Kunming, China). Virus stocks were prepared in 2BS cells and stored in aliquots at −80 °C. Virus titers were determined by standard RT-qPCR as described below. HAV strain HM175/18f (GenBank accession no. M59808) was kindly provided by Prof. Qiang Ding (Tsinghua University, Beijing).
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