For GABAergic differentiation, Neural precursors were seeded at a concentration of 5 × 105 cells/cm2 in 24-well plates and cultured in an induction medium containing DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA), 20 ng/mL of EGF (Peprotech®, East Windsor, NJ, USA) and 20 ng/mL of bFGF (Peprotech®, East Windsor, NJ, USA) for 10 days. The medium was then replaced with a medium composed of DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA) supplemented with 10 ng/mL EGF (Peprotech®, East Windsor, NJ, USA) and 10 ng/mL of all-trans-retinoic acid (Sigma-Aldrich®, St. Louis, MO, USA) which was maintained for 7 days. Finally, the maturation medium containing DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA), 10 ng/mL EGF (Peprotech®, East Windsor, NJ, USA), 5 ng/mL of brain-derived growth factor-BDNF (Peprotech®, East Windsor, NJ, USA) and 1µM of cyclic Di-Buthyril AMP (Sigma-Aldrich®, St. Louis, MO, USA) was added and maintained for 7 days [18 (link)].
Dmem ham f12
Manufactured by Merck Group
Sourced in United States, United Kingdom
DMEM/Ham-F12 is a cell culture medium that provides a balanced solution of nutrients and salts to support the growth and maintenance of various cell types in vitro. It is a combination of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture, which together provide a comprehensive formulation to support a wide range of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (4 mentions)
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Pen strep, by Merck Group (3 mentions)
Dmem ham f 12, by Harvard Bioscience (3 mentions)
β glycerol phosphate, by Merck Group (3 mentions)
Collagenase p, by Merck Group (2 mentions)
Pdgf aa, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Collagen type 1, by Corning (2 mentions)
Forskolin, by Merck Group (2 mentions)
Fungizone, by Calibre Scientific (2 mentions)
Ciprobay, by Fresenius (2 mentions)
Penicillin streptomycin pen strep, by Harvard Bioscience (2 mentions)
Glutamaxtm, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
21 protocols using dmem ham f12
1
GABAergic Differentiation of Neural Precursors
2
Antioxidant and Cytotoxicity Assays
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All the solutions were prepared with ultrapure water (Millipore, São Paulo, Brazil). Folin–Ciocalteu reagent, 2-thiobarbituric acid, 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), iron chloride(III) hexahydrate, copper(II) sulfate pentahydrate, acetonitrile, high-performance liquid chromatography analytical standards (Gallic, syringic, 2-hydroxycinnamic, protocatechuic, 2,4-dihydroxybenzoic, 2,5-dihydroxybenzoic, p-coumaric, 5-O-caffeoylquinic, caffeic, ferulic, rosmarinic, and ellagic acids; quercetin-3-rutinoside, procyanidin A2, (−)-epicatechin, (+)-catechin, and quercetin), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Dulbecco’s modified Eagle’s medium (DMEM)/low glucose, DMEM/Ham-F12, DCFH-DA (dichlorofluorescein diacetate), penicillin, and streptomycin were purchased from Sigma-Aldrich (São Paulo, Brazil). 2,4-Dihydroxybenzoic and 2,5-dihydroxybenzoic acids were purchased from Carl Roth (Karlsruhe, Germany). Ethyl alcohol 99.8%, methyl alcohol 99.8%, petroleum ether, ascorbic acid, and glacial acetic acid were purchased from Pró-Análise (Porto Alegre, Brazil). A549, HCT8, and IMR90 (healthy lung fibroblast cells) cell lines were obtained from the Rio de Janeiro cell bank (BCRJ, Rio de Janeiro, Brazil).
3
Isolation of Pancreatic Stromal Cells
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PSCs and CAFs were isolated as described previously (44 (link), 53 (link)). Briefly, murine pancreata were isolated from WT C57BL/6J mice and digested enzymatically with 0.1% collagenase P (Sigma-Aldrich, Merck KGaA) at 37°C for 25 min. We used a cube of 3 × 3 × 3 mm for isolating CAFs from KPfC or κB-Ras mouse-derived pancreata. After centrifugation with 200g at room temperature for 5 minutes, the homogenized tissue was resuspended in cell culture medium (DMEM/Ham F12 1:1, supplemented with 10% FCS and 1% penicillin/streptomycin; Sigma-Aldrich) and seeded onto FCS-coated tissue culture dishes, glass-bottom dishes, or hydrogel-coated glass-bottom dishes for 2 hours. CAFs derived from KPfC or κB-Ras–deficient mice were allowed to adhere for only 30 minutes, as we found that contamination with cancer cells became substantial after 2 hours. Afterward, nonadherent cells were vigorously washed off the tissue culture plate, resulting in a homogeneous PSC or CAF culture. Depending on the experiments, PSCs and CAFs were cultured for different time periods (2 hours, 24 hours, 3 days, 6 days, 9 days) with pHe6.6 or pHe7.4 media. To avoid trypsin-mediated PSC activation, PSCs and CAFs were used for experiments without passaging directly after isolation (54 (link)).
4
Neural Differentiation of Stem Cells
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Neural precursors were seeded at a concentration of 5 × 104 cells/cm2 and the inducing medium composed of a neurobasal medium (Gibco® BRL, Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10 ng/mL of bFGF (Peprotech®, East Windsor, NJ, USA) and 10 ng/mL EGF (Peprotech®, East Windsor, NJ, USA) was added. Every 3 days of culture, 50% of the medium was replaced with the neurobasal medium supplemented with 10 ng/mL of platelet-derived growth factor—PDGF-AA (Peprotech®, East Windsor, NJ, USA), 10 ng/mL of bFGF (Peprotech ®, East Windsor, NJ, USA) and 10 ng/mL of Sonic Hedgehog (Peprotech®, East Windsor, NJ, USA). After six days of cultivation, the inducing medium was entirely replaced by a fresh inducing medium. Finally, the final differentiation medium was composed of DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA) supplemented with 3% FBS (Sigma-Aldrich®, St. Louis, MO, USA) and 15 nM of F3 (R&D System®, Minneapolis, MN, USA) [20 (link)].
5
Isolation and Culture of Human Umbilical Cord Cells
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Explant Method: HUCs were washed with phosphate buffer saline (PBS) with 3% antibiotic (300 IU/mL penicillin, 0.3 mg/mL streptomycin), the longitudinal intersection was performed, and the vein and arteries were removed. The tissue was fragmented into 2 mm × 2 mm pieces, which were seeded onto culture plates containing DMEM/Ham-F12 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% Fetal bovine serum (FBS) and 1% antibiotic (100 IU/mL penicillin, 0.1 mg/mL streptomycin) and incubated at 37 °C and 5% CO2. The new replacement of the medium was performed after five days of culture. After that, the medium was replaced every 72 h up to 85% of confluence [12 (link)].
6
TNBC Cell Line Culture Protocol
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The studies were conducted on MDA-MB-436 cells—TNBC derived, pleural effusion origin, infiltrating ducal carcinoma and of claudin-low molecular subtype. The cell line was purchased from Cell Lines Service (CLS, Hamburg, Germany) and cells were grown according to the manufacturer′s instructions in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. DMEM: Ham F-12 in a 1:1 ratio supplemented with L-glutamine, penicillin-streptomycin, and 10% fetal bovine serum was used as growth medium (all from Sigma Aldrich, Saint Louis, MO, USA).
7
Isolation of Wharton's Jelly Mesenchymal Stem Cells
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The isolation of WJ-MSC was performed using the explant method. First, the umbilical cord samples were washed with phosphate buffer saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) with 3% antibiotic (300 IU/mL penicillin, 0.3 mg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, USA) to remove blood cells. Next, the cord was cut lengthwise to remove the veins and arteries and was broken into pieces measuring approximately 2 × 2 mm, which were placed onto culture plates and incubated at 37 °C and 5% CO2 for 5 min. After initial incubation, a standard culture medium composed of DMEM/Ham-F12 (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic (100 IU/mL penicillin, 0.1 mg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, USA) was added. The first change to the culture medium was performed 5 days after isolation, and the following changes were performed every 72 h until the cells reached 85% confluence [12 (link),13 (link)].
8
Isolation of Pancreatic Stromal Cells
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PSCs and CAFs were isolated as described previously (44 (link), 53 (link)). Briefly, murine pancreata were isolated from WT C57BL/6J mice and digested enzymatically with 0.1% collagenase P (Sigma-Aldrich, Merck KGaA) at 37°C for 25 min. We used a cube of 3 × 3 × 3 mm for isolating CAFs from KPfC or κB-Ras mouse-derived pancreata. After centrifugation with 200g at room temperature for 5 minutes, the homogenized tissue was resuspended in cell culture medium (DMEM/Ham F12 1:1, supplemented with 10% FCS and 1% penicillin/streptomycin; Sigma-Aldrich) and seeded onto FCS-coated tissue culture dishes, glass-bottom dishes, or hydrogel-coated glass-bottom dishes for 2 hours. CAFs derived from KPfC or κB-Ras–deficient mice were allowed to adhere for only 30 minutes, as we found that contamination with cancer cells became substantial after 2 hours. Afterward, nonadherent cells were vigorously washed off the tissue culture plate, resulting in a homogeneous PSC or CAF culture. Depending on the experiments, PSCs and CAFs were cultured for different time periods (2 hours, 24 hours, 3 days, 6 days, 9 days) with pHe6.6 or pHe7.4 media. To avoid trypsin-mediated PSC activation, PSCs and CAFs were used for experiments without passaging directly after isolation (54 (link)).
9
Culturing hFOB and MSC Cells
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The hFOB 1.19 (ATCC CRL-11372) cell line was cultured in T75 flasks and expanded by subculturing (0.25% Trypsin EDTA, 1:4 slitting ratio). hFOBs were cultured in complete media comprised of DMEM/HAM F12 (Sigma-Aldrich, UK) supplemented with 10% foetal bovine serum (Sigma-Aldrich, UK) and 2 mM L-glutamine (PAA, UK). Cells were used between passages 6 and 10. MSCs were extracted from vertebral bone marrow samples collected by surgeons at the Royal Victoria Hospital, Belfast, during the placement of intervertebral screws. After collection, samples were processed to extract the white blood cell-containing component, which was subsequently cultured until 75% confluent. MSCs were cultured in α-MEM (Thermo Fisher Scientific, UK) supplemented with 10% foetal bovine serum, 2 mM L-glutamine and 100 U/ml pen-strep (all sourced from Thermo Fisher Scientific, UK). After initial growth, MSCs were expanded by subculturing (using 0.25% Trypsin-EDTA and a 1:4 splitting ratio); no cells beyond passage 6 were used in experiments (see Figs 2 and 3 for results using these cells).
10
Isolation and Osteogenic Differentiation of MSCs from Bone Fragments
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Human mesenchymal stem cells (MSCs) loosely attached to cancellous bone structures were collected from bone fragments by washing in phosphate buffered saline (PBS) (Gibco, Darmstadt, Germany) as described before [16 (link)]. Following washing the cells in PBS were collected. After centrifugation, cells were resuspended in Dulbecco’s Medium Essential Medium (DMEM)/Ham F-12 (Biochrom, Berlin, Germany) supplemented with 20% fetal bovine serum (FBS) (Sigma, Taufkirchen, Germany) and 1% penicillin/streptomycin (Pen/Strep) (Biochrom, Berlin, Germany) and seeded at a density of 2 × 106 cells/cm2 in collagen type I (Corning, Bedford, MA, USA) coated flasks. MSCs were expanded by subculturing and further cultivated in osteogenic differentiation medium (ODM) consisting of DMEM/Ham F-12; 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA); 10 mM β-glycerol phosphate (Sigma-Aldrich); 50 μM ascorbate-2-phosphate (Sigma-Aldrich); 10% FBS, and 1% Pen/Strep for 2 weeks to obtain osteogenic lineage cells. Cells were derived from several MSC donors at the age of 69 (m), 53 (f), 70 (f) and 73 (f).
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