The area vitellina of yolk sacs was dissected from the area vasculosa as previously described.5 The intact cell sheets of the area vitellina were disrupted by repeated pipetting until a homogenous cell suspension was obtained. Cells were resuspended in fresh DMEM‐F12 medium (Sigma) supplemented with 10% FCS, 2 mM l ‐glutamine, 0.1 mg/mL streptomycin, and 100 units/mL penicillin, plated at high density on sterile 6‐well cell culture dishes and grown for 48 hours in complete DMEM‐F12 medium at 37°C and 5% CO2. For the detection of SMAD5 phosphorylation, EECs were washed 2× in PBS, starved for 2 hours in Opti‐MEM reduced serum medium followed by the incubation of the cells with recombinant mouse BMP4 or BMP7 proteins (R&D systems) at the indicated concentrations for 40 minutes. For the analysis of gene expression in BMP‐stimulated EECs, freshly isolated EECs were cultured for 48 hours in complete DMEM‐F12 medium containing 200 ng/mL recombinant BMP4 or BMP7 proteins as indicated in the figure legends (medium and BMPs were replaced after 24 hours).
Dmem f12 medium
Manufactured by Merck Group
Sourced in United States, Germany, Poland, Macao, United Kingdom, Sao Tome and Principe, Japan, Italy, Switzerland, India
DMEM/F12 medium is a widely used cell culture medium formulation developed by Dulbecco and Freeman. It is a basal medium that provides a balanced salt solution, amino acids, vitamins, and other nutrients required for the growth and maintenance of a variety of cell types.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (49 mentions)
Fetal bovine serum (fbs), by Merck Group (31 mentions)
Penicillin, by Thermo Fisher Scientific (20 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
Penicillin, by Merck Group (16 mentions)
Penicillin streptomycin, by Merck Group (16 mentions)
Streptomycin, by Merck Group (15 mentions)
L glutamine, by Thermo Fisher Scientific (15 mentions)
Rpmi 1640 medium, by Merck Group (12 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (12 mentions)
Insulin, by Merck Group (11 mentions)
Epidermal growth factor (egf), by Merck Group (11 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (11 mentions)
Glutamax, by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
L glutamine, by Merck Group (7 mentions)
Trypsin, by Merck Group (7 mentions)
Hydrocortisone, by Merck Group (6 mentions)
260 protocols using dmem f12 medium
1
Isolation and BMP Stimulation of Endothelial Cells
2
Isolation and Stimulation of OA Chondrocytes
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Human samples including cartilage and synovial fluid samples were obtained from OA patients undergoing total knee arthroplasty. Tissues and synovial fluids were obtained from 4 OA patients including 1 man and 3 women with mean age of 74 years (59-83 years) who underwent total knee arthroplasty. Obtained cartilage were fixed by 10% formalin for 24 h, decalcified by EDTA for 1 week at 4°C and then embedded frontally in paraffin. To isolate OA chondrocytes, cartilage was cut into small pieces and digested with 0.15% collagenase II solution (Gibco) in DMEM/F12 medium (Sigma) overnight at 37°C-water bath with shaking (Oseni et al., 2013 (link)). Cells were suspended in DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 25 mg/L penicillin/streptomycin and cultured in a 37°C humidified atmosphere containing 5% CO2. After 5 days, chondrocytes were detached using 1% trypsin-EDTA solution (Wako), washed by PBS, seeded at 1X105 and stimulated with recombinant human IL-1β or TNF-α (Peprotech) for 24 h. Synovial fluids of OA knee samples were centrifuged at 2000 x g at 4°C for 10 min to remove cells/debris and preserved at -80°C until further analysis.
3
LUHMES Cell Proliferation and Differentiation
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For proliferation, LUHMES cells (15 (link)) were kept on flasks (Nunc, Thermo Fisher Scientific, Waltham, MA, USA), coated with poly-L-ornithine (0.1 mg/ml; 4°C, overnight; Sigma-Aldrich, St. Louis, MO, USA) in a DMEM/F12 medium (Sigma-Aldrich, St. Louis, MO, USA) with 1% N2-supplement (Thermo Fisher Scientific) and a 0.04 μg/ml basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, CT, USA). For differentiation, the cells were seeded in a differentiation medium at a density of 100,000 cells per cm2 on cell culture dishes coated with poly-L-ornithine (0.1 mg/ml; 4°C, overnight; Sigma-Aldrich), followed by coating with fibronection (5 μg/ml, 37°C, overnight; Sigma-Aldrich). The differentiation medium consisted of a DMEM/F12 medium with 1% N2-supplement, 1-μg/ml tetracycline (Sigma-Aldrich), 0.49-μg/ml dibutyryl cyclic adenosine monophosphate, and a 2-ng/ml glial cell-derived neurotrophic factor (GDNF, R&D Systems, Minneapolis, MN, USA).
4
Differentiation and Maintenance of LUHMES Cells
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For proliferation, LUHMES cells were kept in flasks coated with poly-L-ornithine (0.1 mg/mL; 4 °C, overnight; Sigma-Aldrich) in a DMEM/F12 medium (Sigma-Aldrich) with 1% N2-supplement (Thermo Fisher Scientific, Cat#: 17502048), 2 mM L-Glutamine (Thermo Fisher Scientific), and a 0.04 μg/mL human basic fibroblast growth factor (bFGF, Gibco, Cat#: 13256-029). For differentiation, the cells were seeded in a differentiation medium at a density of 100,000 cells per cm2 in the cell culture dishes coated with poly-L-ornithine (50 μg/mL; 4 °C, overnight; Sigma-Aldrich), followed by coating with human fibronectin (1 μg/mL, 37 °C, overnight; Sigma-Aldrich). The differentiation medium consisted of a DMEM/F12 medium with 1% N2-supplement, 1 μg/mL tetracycline (Sigma-Aldrich), 1 mM dibutyryl cAMP (Sigma-Aldrich), and a 2 ng/mL glial cell-derived neurotrophic factor (GDNF) (Sigma-Aldrich, Cat#: G1777). To handle LUHMES spheroid culture, between 1000 and 3000 cells were seeded into ultra-low attachment, U-shaped, 96-well plate (CellCarrier Spheroid ULA 96-well Microplates, Perkin Elmer, Cat#: 6055330) with 100 μL of LUHMES proliferating medium.
5
Culturing Breast Cancer and Mammary Cells
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The MDA-MB-231 (HTB-26) and MCF-7 breast cancer cell lines (American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland). The cancer cell media were supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) and 1.0% penicillin/streptomycin (Lonza, Basel, Switzerland). Conversely, the normal epithelial MCF-12F (CRL-10783) mammary gland cells (ATCC, Manassas, VA, USA) were grown in DMEM/F-12 medium (Sigma-Aldrich, St Louis, MO, USA). The DMEM/F-12 medium was enriched with 7.0% fetal horse serum, 10 µg/mL human insulin, 20 ng/mL epidermal growth factor, 500 ng/mL hydrocortisone, 100 mg/mL cholera toxin, and 1.0% penicillin/streptomycin, all obtained from Sigma-Aldrich, St Louis, MO, USA.
6
Dissociation of Ipsilateral DRG Neurons
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L4–6 DRG neurons from the ipsilateral side of the operation were dissociated using enzyme digestion as previously described (Zhang et al., 2017 (link)). The drug-intervention group DRGs were treated with 17β-estradiol and Cltx. Briefly, the excised ganglia were freed from their connective tissue sheaths and cut into pieces with a pair of sclerotic scissors in DMEM/F12 medium (GIBCO; Thermo Fisher Scientific, Waltham, MA, United States) under low temperature on ice. The fragments were transferred into 5 mL of DMEM/F12 medium containing trypsin (0.4 mg/mL, Sigma) and collagenase (type IA, 0.6 mg/mL, Sigma) and incubated for 5 min at 37°C. The ganglia were then gently triturated using fine fire-polished Pasteur pipettes. The suspension was dissociated in DMEM/F12 medium, supplemented with 10% fetal bovine serum, and DRG neurons were plated on glass cover slips coated with Poly-L-Lysine (Sigma). Cells were maintained in a humidified atmosphere (5% CO2, 37°C) and used for electrophysiological recordings 6–24 h after plating.
7
Xenograft and Sphere Culture of Murine and Human Glioma Cells
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Mice All mouse strains were housed in a barrier facility under protocols approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine. Rag -/-mice were purchased from the Jackson Laboratories (JAX stock #002216), bred at the institution's animal facility, and used for graft or xenograft between 8 and 12 weeks of age.
Cell culture Sphere culture of iEIP cells was established and maintained in serum-free DMEM/F12 medium (Sigma), containing ITS (Invitrogen), epidermal growth factor (EGF) (20 ng/ml, Peprotech), and basic fibroblast growth factor (bFGF) (20 ng/ml; Peprotech) as previously described (60) . The human glioma initiating cells (GS7-11, GS8-11, GSC280) were maintained in serum-free DMEM/F12 medium (Sigma), containing B27 (Invitrogen), 20 ng/ml EGF, and 20 ng/ml bFGF.
Cell culture Sphere culture of iEIP cells was established and maintained in serum-free DMEM/F12 medium (Sigma), containing ITS (Invitrogen), epidermal growth factor (EGF) (20 ng/ml, Peprotech), and basic fibroblast growth factor (bFGF) (20 ng/ml; Peprotech) as previously described (60) . The human glioma initiating cells (GS7-11, GS8-11, GSC280) were maintained in serum-free DMEM/F12 medium (Sigma), containing B27 (Invitrogen), 20 ng/ml EGF, and 20 ng/ml bFGF.
8
Establishing and Maintaining iEIP Cell Spheres
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Sphere culture of iEIP cells was established and maintained in serum-free DMEM/F12 medium (Sigma), containing ITS (Invitrogen), EGF (20 ng/mL, Peprotech), and basic fibroblast growth factor (bFGF; 20 ng/mL; Peprotech) as previously described (17) . The human glioma initiating cells (GS7-11, GSC280) were provided by Dr. Erik Sulman (NYU Langone Health, New York, NY) and maintained in serum-free DMEM/F12 medium (Sigma), containing B27 (Invitrogen), 20 ng/mL EGF, and 20 ng/mL bFGF. Low passage cultures were frozen down and used within 2-3 passages when thawed, for all experiments. All cultures were routinely subject to Mycoplasma testing and authentication.
9
Establishing Human Glioblastoma Stem Cells
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The human glioblastoma cell lines A172 and T98G were obtained from ATCC. A172 and T98G cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Nichirei) and 1% penicillin/streptomycin/amphotericin (Nacalai Tesque, Kyoto, Japan).
To establish human GSCs, samples from human GBMs were dissociated with the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA). After filtration, dissociated cells were cultured in serum-free medium with growth factors as previously described (Anai et al., 2014 (link); Singh et al., 2004 (link)). All cells were incubated at 37 °C in an atmosphere containing 5% CO2. To confirm their tumor-forming potential, 1 × 105 GSCs were injected into the brains of 8-week-old female ICR-nu mice (Crlj:CD1-Foxn1nu; Charles River Japan) after anesthesia. Four weeks later, xenograft tumors had formed in the brain. Pathologically, the tumors showed high cellularity and necrosis similar to that of the original tumor (Fig. 3 A and B).
To establish human GSCs, samples from human GBMs were dissociated with the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA). After filtration, dissociated cells were cultured in serum-free medium with growth factors as previously described (Anai et al., 2014 (link); Singh et al., 2004 (link)). All cells were incubated at 37 °C in an atmosphere containing 5% CO2. To confirm their tumor-forming potential, 1 × 105 GSCs were injected into the brains of 8-week-old female ICR-nu mice (Crlj:CD1-Foxn1nu; Charles River Japan) after anesthesia. Four weeks later, xenograft tumors had formed in the brain. Pathologically, the tumors showed high cellularity and necrosis similar to that of the original tumor (
10
Culturing Pancreatic Cancer and Stellate Cells
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All pancreatic cells were maintained free of antibiotics in DMEM:F12 medium (Sigma, Gillingham, UK) supplemented with 10% foetal bovine serum (FBS; Gibco, Loughborough, UK) at 37 °C in 5% CO2. Human Miapaca2 and Panc‐1 pancreatic cancer cell lines were a kind gift from Professor Hemant Kocher (Barts Cancer Institute, London, UK). Mouse pancreatic cancer cell lines R254 and DT6066 were derived from KPF and KPC pancreatic tumours, respectively [8 (link), 11 (link)], and were a kind gift from Professor Kairbaan Hodivala‐Dilke (Barts Cancer Institute). PS1 stellate cells were a kind gift from Professor Hemant Kocher (Barts Cancer Institute). Mouse PSCs were isolated from wildtype C57BL/6 mice as described [8 (link)]. The 1089 myoepithelial cell line, a kind gift from Professor Louise Jones (Barts Cancer Institute), was maintained free of antibiotics in Ham's F12 medium (Gibco), supplemented with 10% FBS, 0.5 μg/ml hydrocortisone (Sigma), 10 ng/ml Epidermal Growth Factor (Sigma), and 5 μg/ml Insulin (Sigma).
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