U2OS cells were maintained in McCoy’s 5A medium (Gibco) including GlutaMAX (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/ml each of penicillin and streptomycin (Lonza). PC12-tetOFF cells were maintained in low glucose DMEM medium (1 g/L, Lonza), supplemented with 10% FBS, 5% horse serum (HS, Gibco), 2 mM GlutaMAX (Gibco) and 100 U/ml each of penicillin and streptomycin. PC12 cells were grown on poly-L-lysine (Sigma) coated surfaces for maintenance and experiments; U2OS cells were grown on poly-L-lysine surfaces only for experiments. All cells were grown at 37 °C and an atmosphere of 5% CO2.
Dmem low glucose medium
Manufactured by Lonza
Sourced in United States
DMEM low glucose medium is a cell culture medium that provides essential nutrients for the growth and maintenance of cells in vitro. It contains a lower concentration of glucose compared to standard DMEM, which is suitable for cell lines that are sensitive to high glucose levels.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Lonza (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Lonza (2 mentions)
Penicillin, by Merck Group (2 mentions)
Co2 incubator, by Sartorius (2 mentions)
Pen strep, by Lonza (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Hepes buffer, by Lonza (1 mentions)
Nct 503, by Merck Group (1 mentions)
Cb 839, by MedChemExpress (1 mentions)
Mda mb 468 breast cancer cell line, by American Type Culture Collection (1 mentions)
Egm 2, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
8 protocols using dmem low glucose medium
1
Cell Culture Maintenance Protocols
2
Tumor Cell Culture Protocols
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Human breast cancer T47D, MCF-7 and MDA-MB-468 cells and mouse colon cancer CT-26 cells were from the Korean Cell Line Bank. Breast cancer cells were maintained in RPMI 1640 medium and CT-26 cells in low-glucose DMEM medium (Lonza, Switzerland), supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/L streptomycin at 5% CO2 in 37°C. Experiments were performed 2 or 3 days following seeding, when cell confluence was approximately 90%.
Cells were alkalinized or acidified with pH buffers prepared from Hank's balanced salt solution (Gibco Invitrogen) containing 0.2% bovine serum albumin and 1 g/L glucose. Solutions at pH 6.2 and pH 6.8 were buffered with 10 mM MES (Sigma); solutions at pH 7.2, 7.4 and pH 7.8 were buffered with 10 mM HEPES (Sigma). Solution pH was titrated immediately before use.
Cells were alkalinized or acidified with pH buffers prepared from Hank's balanced salt solution (Gibco Invitrogen) containing 0.2% bovine serum albumin and 1 g/L glucose. Solutions at pH 6.2 and pH 6.8 were buffered with 10 mM MES (Sigma); solutions at pH 7.2, 7.4 and pH 7.8 were buffered with 10 mM HEPES (Sigma). Solution pH was titrated immediately before use.
3
Isolation and Culture of Human MSCs
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Human bone marrow-derived MSCs were obtained from the Inbiobank Stem Cell Bank (http://www.inbiomed.org/Index.php/servicios_externos/inbiobank ) as described previously [35 ]. In short, cadaveric marrow was obtained from brain-dead donors after informed consent and under the Spanish National Organization of Transplant supervision (ONT). MSCs were positive for CD29, CD73, CD90, CD105, CD166 and CD146 but negative for markers of the hematopoietic lineage; CD34, CD45, CD14, CD19 and CD31. Moreover, they displayed a fibroblast-like phenotype and showed at least a tri-lineage potential differentiating into osteocytes, chondrocytes and adipocytes. MSCs were cultured in DMEM low-glucose medium supplemented with 10% FBS (Lonza, Walkersville, MD, USA), 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO, USA). Upon reaching confluence MSCs were treated with 0.25% Trypsin-EDTA solution (Sigma) and seeded at a density of 1000–1500 MSC/cm2. Cells were obtained from the Inbiobank Stem Cell Bank at passage three and all experiments were carried out with cells from low passages (passage number 4–8).
4
Establishment and Culture of Burkitt Lymphoma Cell Lines
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All human Burkitt lymphoma cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). MDA-MB-468 breast cancer cell line was purchased from American Type Culture Collection (ATCC). BL cell lines were grown in RPMI-1640 medium supplemented with heat-inactivated 10% or 20% (RAMOS) fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin, and 25 mmol/L HEPES buffer (all from Lonza) at a density of 0.5–2.0 × 106 cells/mL. MDA-MB-468 cell line was cultured in DMEM low glucose medium (Lonza) supplemented with FBS, penicillin, and streptomycin as above. All cells were grown in a humidified atmosphere at 37 °C with 5% CO2.
PHGDH inhibitor NCT-503 (N-(4,6-dimethylpyridin-2-yl)-4-(4-(trifluoromethyl)benzyl) piperazine-1-carbothioamide) and NCT-503 inactive control (N-(4,6-dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide), unable to inhibit PHGDH) were purchased from Sigma-Aldrich. Glutaminase inhibitor CB-839 was purchased from MedChemExpress. All compounds were diluted in sterile dimethyl sulfoxide (DMSO) to obtain 10 mM stocks, aliquoted, and stored in 2–8 °C according to manufacturer’s recommendations.
PHGDH inhibitor NCT-503 (N-(4,6-dimethylpyridin-2-yl)-4-(4-(trifluoromethyl)benzyl) piperazine-1-carbothioamide) and NCT-503 inactive control (N-(4,6-dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide), unable to inhibit PHGDH) were purchased from Sigma-Aldrich. Glutaminase inhibitor CB-839 was purchased from MedChemExpress. All compounds were diluted in sterile dimethyl sulfoxide (DMSO) to obtain 10 mM stocks, aliquoted, and stored in 2–8 °C according to manufacturer’s recommendations.
5
Endometrial Cell Culture Protocol
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Endometrial tissue samples (n = 2) were washed with phosphate buffer saline (PBS), minced into tiny pieces then digested with 1 mg/ml type 1A collagenase (Gibco, life technologies, USA) for 60 min at 37 °C. Afterwards, cells and cell aggregates were filtered through 100 μm pore size cell strainer (Greiner Bio-one, Germany). Obtained cell suspensions were centrifuged, resuspended and cultured in DMEM low glucose medium (Lonza, Belgium) supplemented with 10% FBS, 100 units/ml penicillin (Gibco), 100 μg/ml streptomycin (Pen-Strep, Lonza) and 2 mM/L glutamax (Gibco) [11] (link). Two cell cultures were obtained, each from a separate endometrial biopsy. Cultured cells were then incubated at 37 °C and humidified atmosphere with 5% CO2 concentration in a CO2 incubator (Sartorius stedim biotech, GmbH, Germany). Media was exchanged every 2–3 days. Subsequent subculture was done at approximately 80% confluence till passage 3.
6
Endothelial Cell Responses to Cigarette Smoke and Oxidative Stress
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Cigarette smoke condensate (CSC) was purchased by Murty Pharmaceuticals (Lexington, KY) (stock solution 40 mg/mL in DMSO) and diluted in cell medium at the final concentration of 20 μg/mL, based on previous studies.21 , 22 , 23 Human umbilical vein endothelial cells (HUVECs) (Lonza, Monza, Italy) were grown in EGM2 (Lonza) complete medium in a humidified incubator at 37°C and 5% CO2. For CSC and H2O2 experiments, HUVECs were serum starved for 4 hours using DMEM low‐glucose medium (Lonza). The medium was then changed with DMEM low‐glucose medium containing 20 μg/mL CSC or 100 μmol/L H2O2. Cells incubated in DMEM low‐glucose medium with vehicle (DMSO <0.01%) were used as controls. HUVECs were harvested after 15 minutes, 30 minutes, 1 hour, 4 hours, and 12 hours of treatment (t=0). Cells between passages 2 and 4 were used for all the experiments.
7
Endometrial Mononuclear Cell Isolation and Culture
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As previously reported (Kao et al., 2011[30 (link)]), the collected endometrial tissue samples were enzymatically digested to drive the tissue mononuclear cells which were cultured in DMEM low glucose medium (Lonza) supplemented with 10 % FBS, 100 units/ml penicillin (Gibco), 100 μg/ml streptomycin (Pen-Strep, Lonza) and 2 mM/L glutamax (Gibco). The cultures were then incubated in humidified atmosphere with 5 % CO2 concentration in CO2 incubator (Sartorius Stedim Biotech, GmbH, Germany). Media exchange was done every 2-3 days. After attaining confluence, the cultures underwent subsequent passaging until passage 3.
8
Culturing Cancer Cell Lines on PMMA Films
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