MCF7 breast cancer cells were obtained from ATCC and cultured in DMEM (Sigma Aldrich). For hypoxic stimulation MCF7 cells were cultured in low glucose DMEM (Sigma Aldrich) in a multi-gas N2/CO2 hypoxic chamber at 1 % pO2; parallel, MCF7 cells were cultured in low glucose DMEM at 21 % O2 to serve as a normoxic control.
Dmem low glucose
Manufactured by Merck Group
Sourced in United States, Germany, Japan, United Kingdom, Switzerland
DMEM low glucose is a cell culture medium formulated with a reduced concentration of glucose compared to standard DMEM. It is designed to support the growth and maintenance of cells that have a lower glucose requirement or are sensitive to high glucose levels.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions)
Penicillin streptomycin, by Merck Group (18 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (18 mentions)
Fetal bovine serum (fbs), by Merck Group (17 mentions)
Dexamethasone (dex), by Merck Group (15 mentions)
Glutamax, by Thermo Fisher Scientific (15 mentions)
Dmem high glucose, by Merck Group (14 mentions)
Penicillin, by Merck Group (11 mentions)
Streptomycin, by Merck Group (11 mentions)
L glutamine, by Merck Group (9 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Antibiotic antimycotic mixture, by Euroclone (7 mentions)
L glutamine, by Euroclone (7 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (6 mentions)
Axiovert 40 microscope, by Zeiss (6 mentions)
Moticam 2300 camera, by Motic (6 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Rpmi 1640 medium, by Merck Group (5 mentions)
Ascorbic acid, by Merck Group (5 mentions)
165 protocols using dmem low glucose
1
Hypoxic Stimulation of MCF7 Cells
2
Osteogenic and Adipogenic Differentiation of hTSCs
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hTSCs were seeded at a concentration of 3 × 104 cells/cm2 in a growth medium, and after 24 hours, cells were switched to an osteogenic or adipogenic medium for 17 days or 21 days, respectively. Osteogenic differentiation was obtained by culturing cells in the presence of DMEM-low glucose (Merck) supplemented with 4 mM L-glutamine (Euroclone), 1% antibiotic-antimycotic mixture (Euroclone), 10% FBS (HyClone, Thermo Fisher Scientific), 10 nM cholecalciferol (Merck Millipore), and the mesenchymal stem cell osteogenesis kit (Merck Millipore) according to the manufacturer's instructions. Adipogenic differentiation was induced by culturing cells in the presence of DMEM-low glucose supplemented with 4 mM L-glutamine, 1% antibiotic-antimycotic mixture, 10% FBS, and the mesenchymal stem cell adipogenesis kit (Merck Millipore), according to the manufacturer's instructions. To evaluate the effects of ganglioside GM1 treatment (Santa Cruz Biotechnology) on differentiation, hTSCs were cultured for 17 days in an osteogenic medium or 21 days in adipogenic medium supplemented with 1, 10, 50, and 100 μM GM1. To evaluate the effects of the platelet-derived growth factor-BB (PDGF-BB, Thermo Fisher Scientific) on osteogenic differentiation, cells were cultured in an osteogenic medium containing PDGF-BB at the final concentration of 10 ng/ml. The differentiation medium was changed every 2-3 days.
3
Murine Hepatocyte Isolation and Manipulation
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The hepatocytes were isolated from male 8-week-old C57/B6J mice fasted for 6 h. The viability of freshly isolated cells was examined using trypan blue (Sigma, MO, USA) ensuring more than 90% viability. After the hepatocytes were attached to dishes in DMEM-low glucose (Sigma) plus 10% (vol/vol) FBS (Gibco, ON, USA) for 4 h, the medium was changed to DMEM-low glucose containing 0.1% bovine serum albumin (BSA) (wt/vol) (Sigma) for further studies on the same day. The hepatocytes were incubated with the control, overexpressed Smad3 (ViGene, Shandong, China), overexpressed Ets-1, shNC and shEts-1 adenovirus with 50 MOI or transduced with the negative control, Smad4 and Smad2/3-specific small interfering RNA (siRNA) (RiboBio, Guangdong, China) using Lipofectamine 2000 (Invitrogen, NY, USA). The siRNA sequences used for analysis are listed in Supplementary Table S1 .
4
Isolation and Culture of Rat Mesenchymal Stem Cells
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The mesenchymal stem cells were isolated from femurs and tibiae of male Wistar rats ranging in weight from 150-250 g. For the separation of the mononuclear fraction of bone marrow, the material was subjected to the Histopaque protocol (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 1800 rpm for 30 minutes, the mononuclear cells were selected from the interface established between the Histopaque and PBS, and were washed with sterile PBS. These cells were then cultured in 25 cm2 polystyrene flasks with 5 mL of DMEM low glucose (Dulbecco's Modified Eagle's medium, DMEM low glucose - Sigma Chemical Company, St. Louis, USA), supplemented with 10% fetal bovine serum (FBS), and maintained at 37°C, in a humidified 5% (v/v) CO2 in air atmosphere for 10 to 15 days. For each cell culture passage, the cells were subjected to trypsin-EDTA (Sigma Chemical Company, St. Louis, USA), seeded, and cultured until to obtain adequate cell number for the experiments.
5
Mesenchymal Differentiation Capacity Evaluation
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To confirm the mesenchymal activity of those cells that grew out naturally from the lipoaspirate clusters, while under culture conditions, their in vitro differentiation capacity was studied according to Noël et al. (26) . Briefly, 10 × 10 3 cells/ cm 2 were cultured in DMEM-low glucose supplemented with 10% FBS, 0.5 mM isobutyl-methyl xanthine (IBMX; Sigma-Aldrich), 200 μM indomethacin, 1 μM dexamethasone, and 10 μg/ml insulin (all from Sigma-Aldrich); after 2 weeks they were stained with fresh Oil red O solution (Sigma-Aldrich) for adipogenesis. After 3 weeks of culture in DMEM-low glucose supplemented with 10% FBS, 10 mM b-glycerophosphate, 0.2 mM ascorbic acid, and 10 nM dexamethasone (all from Sigma-Aldrich), for osteogenic differentiation, cells were stained with calcein solution (Sigma-Aldrich) to evidence mineralization (16) and with 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma-Aldrich) to evidence alkaline phosphatase activity (8). To demonstrate chondrogenic differentiation, pellets of 5 × 10 5 cells were cultured for 3 weeks in chondrogenic medium (Lonza, Cologne, Germany), formalin-fixed, embedded in paraffin, and immunostained for type II collagen by incubation with rabbit polyclonal anti-collagen type II primary antibody 1 mg/ml (Biogenesis, Oxford, UK) and with secondary antibody conjugated to peroxidase (Vector Laboratories).
6
Culture of PC-12 cells expressing EKAR2G1
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PC‐12 cells stably expressing the EKAR2G1 construct, described earlier in Ryu et al (2015 ), and PC‐12 Neuroscreen‐1 (NS‐1, gift from Tobias Meyer) were cultured using low‐glucose DMEM (Sigma) supplemented with 10% horse serum (HS; Sigma), 5% fetal bovine serum (FBS; Sigma), and 1% penicillin/streptomycin. Cells were cultured on plastic tissue culture dishes (TPP) coated with 50 μg/ml collagen from bovine skin (Sigma). Cells were passaged at 70% confluence by detaching cells using a cell scraper (Fisher).
7
CGA-Induced Osteoblast Differentiation
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To induce osteoblast differentiation, 1 × 104 rat BMSCs were seeded in 24-well plates containing osteoblast differentiation medium (ODM; low-glucose DMEM supplemented with 10% FBS, 10−8 M-dexamethasone,10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid; all from Sigma-Aldrich, Shanghai, China). Cells exposed to ODM without CGA treatment were used as a positive control. Cells exposed to standard medium without differentiation factors were treated with various concentrations of CGA (Sigma-Aldrich), ranging from 0 μM (control) to 10 μM. All experiments were performed in triplicate.
8
Multilineage Differentiation Protocols for Stem Cells
9
Photocrosslinked GelMA Scaffolds for Cell Culture
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Cellular and acellular scaffolds of 10% GelMA were cast and photocrosslinked, both with and without transglutaminase as described above. After photocrosslinking, scaffolds were incubated in culture media containing low glucose DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco), 2 mM L-glutamine (Gibco), and 15 mM HEPES (Gibco), 20 ng/ml epidermal growth factor (EGF) and 1 ng/ml fibroblast growth factor (FGF) (R&D Systems Inc, Minneapolis, MN, USA). At days 1, 3, and 7, a biological triplicate of each condition was removed to perform a metabolic activity assay using CellTiter-Blue® Reagent (Promega, Madison, WI, USA) according to manufacturer's instructions and based on a previously published method which assessed metabolic activity of cells in 3D (42 (link)). Scaffolds were incubated for 3 h in a 1:5 ratio of media and reagent. After incubation, supernatant was collected and measured using a CLARIOstar plate reader at 550–15 nm excitation and 600–20 nm emission using the same gain for all readings. After the metabolic assay, scaffolds were rinsed in PBS before performing a mechanical unconfined compression test as described.
10
Isolation and Characterization of Mouse Mesenchymal Stem Cells
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The isolation of mouse MSCs from the bone marrow of Balb/c mice and characterization of MSCs were identified with our previously reported method [10 (link)–13 (link)]. Mouse MSCs were then cultured in low-glucose DMEM (Sigma-Aldrich) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). The quality and purity of obtained MSCs were assessed, and the isolated MSCs were able to be differentiated into the bone, adipose tissue, and hepatocyte in vitro (see Additional file 1 ) [10 (link)–13 (link)]. MSCs were used at passages 8–13 in this study. The study was approved by the Medical and Ethics Committee of our hospital (approval no. 201701193B0).
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