EHTs were produced as previously explained [8 (link),16 (link)], and the experimental design is described in Figure 1 B. Briefly, iPSC-CMs at day 14 were mechanically selected from the differentiation plate and digested with 5 mg/mL of collagenase type 1 (Worthington Biochemical Corporation, Lakewood, NJ, USA) for 45 min at 37 °C under agitation and later with 2 mg/mL of collagenase type 2 (Worthington Biochemical Corporation) for 45 min at 37 °C. Dissociated iPSC-CMs were counted (~1–2 × 106 cells) and combined with 5 mg/mL of bovine fibrinogen (Merck, Darmstadt, Germany), 100 μL/mL of Matrigel (BD), DMEM (2 × 1 g/L D-Glucose, Biochrom), and 0.25 U/mL of thrombin (Merck), pipetted into the 2% agarose (Thermofisher Scientific) molds previously solidified in a 24-well culture dish with the silicone posts racks. After 90 min at 37 °C and 7% CO2, 300 μL of the cell culture medium was added to easily remove the EHTs, and they were transferred into a new 24-well plate. EHTs were maintained at 37 °C in a 7% CO2 humidified cell culture incubator with media changes three times a week and functional measurements twice a week. The EHT medium consisted of DMEM (Biochrom, Cambridge, UK), 10% inactivated horse serum (Merck), 1% penicillin/streptomycin (Merck), 10 μg/mL of insulin (Merck), and 33 μg/mL of aprotinin (Merck).
Dulbecco modified eagle medium (dmem)
Manufactured by Harvard Bioscience
Sourced in Germany, United Kingdom, United States
DMEM (Dulbecco's Modified Eagle's Medium) is a commonly used cell culture medium that provides essential nutrients and growth factors to support the growth and maintenance of a wide range of cell types. It is a complex, chemically defined medium that contains essential amino acids, vitamins, inorganic salts, and other components required for cell survival and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Harvard Bioscience (52 mentions)
Penicillin streptomycin, by Harvard Bioscience (24 mentions)
Streptomycin, by Harvard Bioscience (20 mentions)
Penicillin, by Harvard Bioscience (19 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (19 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
L glutamine, by Harvard Bioscience (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (10 mentions)
Trypsin, by Harvard Bioscience (8 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (8 mentions)
Rpmi 1640, by Harvard Bioscience (7 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Insulin, by Merck Group (5 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Roswell park memorial institute (rpmi), by Harvard Bioscience (4 mentions)
203 protocols using dulbecco modified eagle medium (dmem)
1
Engineered Human Cardiac Tissues from iPSCs
2
Isolation and Culture of Intestinal Mesenchymal Cells
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For isolation of IMCs, colons from 5–8-wk-old mice were removed, cut in 0.5–1 mm pieces and washed with HBSS (Invitrogen). Intestinal pieces were then incubated in HBSS containing 5 mM EDTA and 1 mM DTT for 20 min at 37°C to remove the epithelial layer. This was followed by incubation with 300 U/ml Collagenase XI (Sigma-Aldrich) and 0.1 mg/ml Dispase (Roche) in DMEM (Biochrom) for 60 min at 37°C. Supernatants were centrifuged and cell pellets were resuspended in DMEM supplemented with 10% FBS (Biochrom), 1% nonessential amino acids (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml amphotericin B (Sigma-Aldrich) and plated in cell culture flasks. At passages 3–4, cells were treated with 10 ng/ml IL-1β (PeproTech) or 1 µg/ml LPS (Sigma-Aldrich) or 10 ng/ml TNF (provided by C. Libert, Ghent University, Belgium) for the indicated time points. For experiments with conditioned medium (CM), cells were plated, serum starved, and then induced with LPS (1 µg/ml) for 8 h. Cells were then washed with PBS and serum-free DMEM and fresh DMEM was added for 48 h. The supernatant was then collected, passed through a 0.2-µm pore size filter, and stored at −80°C. The CM was added on HT-29 epithelial cells for 15 min in a ratio of 1:1 with serum-free DMEM.
3
Culturing Diverse Cell Lines for Experiments
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HEK293T, MCF7, and C2C12 cells were grown in DMEM (Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) (PAA Laboratories) at 37°C and 10% (C2C12) or 5% CO2. Immortalized human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. hFOBs (1.19) were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% FCS, 2 mM l -glutamine, penicillin (100 U/ml)/streptomycin (10 µg/ml), and 0.3 mg/ml G418 (Biochrom AG) at 34°C with 5% CO2 to keep them in a proliferative state. HUVECs were a kind gift from M. Lorenz and V. Stangl (Charité Universitätsmedizin, Berlin, Germany) and cultured on gelatin-coated tissue culture ware in M199 medium supplemented with 20% FCS, 50 µg/ml endothelial cell growth supplement (Corning), 25 µg/ml heparin, 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. HUVECs were used at passage 3 in all experiments. Unless stated otherwise, all cells were starved for 5 h prior to stimulation with their respective growth medium, without FCS supplement, containing 2 mM l -glutamine and penicillin/streptomycin.
4
Cultivation of Human and Monkey Cell Lines for Viral Research
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Human neuroepithelioma cell line SK-N-MC (ATTC HTB-10, ATCC, Manassas, Virginia, USA), and African green monkey kidney Vero cell line (ATTC CCL-81, ATCC, Manassas, Virginia, USA) were obtained from the American Type Culture Collection. Stable transfected SK-N-MC amyloid precursor protein (SK-APP-D1, these cells were generated from SK-N-MC cells (ATCC HTB10, Manassas, Virginia, USA) modified to stably expressing the human isoform APP695 and these cells were kindly provided by Dra. MJ. Bullido (Centro Biología Molecular Severo Ochoa, CBMSO, Madrid, Spain).
SK-N-MC and SK-N-MC APP-D1 cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS); Vero cell line was grown in DMEM supplemented with 5% FBS. All culture mediums contain 1% L-glutamine, and an antibiotic mix (125 μg/mL ampicillin, 125 μg/mL cloxacillin and 40 μg/mL gentamicin); (Sigma, St. Louis, Missouri, USA). All cell lines were cultivated in 5% CO2 at 37 °C.
HSV-1 strain Kos 1.1 was kindly provided by Dra. MJ. Bullido and expanded on the Vero cell line, titrated by plaque assay and stored at −80 °C.
SK-N-MC and SK-N-MC APP-D1 cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS); Vero cell line was grown in DMEM supplemented with 5% FBS. All culture mediums contain 1% L-glutamine, and an antibiotic mix (125 μg/mL ampicillin, 125 μg/mL cloxacillin and 40 μg/mL gentamicin); (Sigma, St. Louis, Missouri, USA). All cell lines were cultivated in 5% CO2 at 37 °C.
HSV-1 strain Kos 1.1 was kindly provided by Dra. MJ. Bullido and expanded on the Vero cell line, titrated by plaque assay and stored at −80 °C.
5
Myoblast Differentiation and Treatment
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The mouse myoblast cell line C2C12 (European Collection of Animal Cell Cultures (ECACC), Salisbury, UK) was propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (all reagents from Biochrom, Berlin, Germany). Myoblasts were differentiated to polynucleated and cell cycle-arrested myotubes as described (Takacs et al., 2012 (link)). Briefly, at 24 hours after seeding of myoblasts, myogenic differentiation was induced by incubation in DMEM, 2% horse serum (Biochrom) and antibiotics as above. After 6 – 7 days of differentiation and one day before infection, the medium was replaced by DMEM, 10% FCS and antibiotics as above. In parallel, proliferating myoblasts were seeded in the same medium. For metabolome analyses, myotubes and myoblasts were cultured in glucose-free DMEM supplemented with 1 g/L glucose, 10% FCS and antibiotics. In some experiments, myoblasts and myotubes were treated with 5 – 10 mM N-acetyl cysteine (NAC), 20 – 40 µM tert-butyl peroxide (Luperox) or with vehicle (mock), starting at 1 hour prior to infection (all reagents from Sigma-Aldrich, Taufkirchen, Germany).
6
Generating ERp57 Knockout C28/I2 Cells
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C28/I2 cells [23 (link)] were kept in DMEM (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1% sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (complete DMEM) at 37°C, 5% CO2, and 100% humidity. To generate ERp57 knockout C28/I2 cells (C28/I2 KO cells), a cotransfection with an ERp57 CRISPR/Cas9 KO plasmid with a Green Fluorescent Protein (GFP) gene and an ERp57 homology directed repair (HDR) plasmid with puromycin resistance and Red Fluorescent Protein (RFP) genes (both from Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) was performed according to the protocol of the manufacturer.
7
Culturing C28/I2 Wildtype and ERp57 KO Cells
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C28/I2 wildtype cells (WT) (Finger et al., 2003 (link)) were cultured in DMEM (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1% sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (DMEM complete) at 37°C, 5% CO2, and 100% humidity. Similar conditions were used for C28/I2 ERp57 knockout cells (KO), generated with CRISPR/Cas9 technology (Rellmann et al., 2019 (link)).
8
3T3 Collagen Contraction Assay
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3T3 CF were reversely transfected with respective siRNAs and after 24 hours of transfection collagen gels were prepared. Gels containing 1 mg/ml Cultrex® 3-D Culture Matrix Rat Collagen I (Trevigen, Gaithersburg, MD, USA), 10 X DMEM (Biochrom, Berlin, Germany) and 1 * 106 of trypsinized 3T3 CF, resuspended in DMEM high glucose +10% FBS were mixed on ice. Before the cells were added to the mix, pH of DMEM was adjusted with 2 M NaOH until the color of DMEM changed from yellow to pink. The collagen-cell suspension was then plated into low attachment 24 well plate (Greiner bio-one, Kremsmünster, Austria). After 4 hours of incubation at 37 °C the edges of the gel were loosened from the borders of the plate with a pipette tip. 500 μl of DMEM +2% FBS was then added on top of the gel. For TGF-β1 stimulation 10 nM mouse TGF-β1 was added. After 48 hours the gels were photographed with a digital camera and the area of the contracted gels was measured by using ImageJ (1.47 V, NIH) software.
9
Chondrocyte Culture and ERp57 Knockout
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C28/I2 WT chondrocytes [53 (link)] were cultured at 37 °C, 5% CO2, and 100% humidity in DMEM (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1% sodium pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin (DMEM complete). ERp57 knockout C28/I2 cells (C28/I2 KO) were generated using CRISPR/Cas9 as previously described [50 (link)] and cultured likewise.
10
Fibroblast-Seeded Collagen Matrix Formation
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Fibroblasts were harvested by trypsinization and counted automatically with the NucleoCounter (Chemometec). After centrifugation at 1200 rpm for 5 min, the cells were resuspended in culture medium to a final concentration of 1 x 106 cells/ml. All ingredients for the collagen matrix were stored on ice before the construction was started and mixed in the exact order listed in the following example for the assembly of at least 6 layers: 1040 μl 10x DMEM (Biochrom), 208 μl 1 M NaOH (Merck), 4,4 ml dH2O (ChemSolute), 260 μl 7.5% NaHCO3 (Carl Roth), 2.6 ml fibroblast growth medium, 4.42 ml (9 mg/ml) rat tail collagen I (Corning; final gel concentration of 2.25 mg/ml). The pH of the solution was adapted to 7.4 with 1 M NaOH and 3.25 ml cell suspension (with a final concentration of 5 x 105 cells/layer) was finally added. After gentle trituration, 2.5 ml solution per layer was transferred to a 6 well culture plate. The collagen polymerized by incubation at 37°C in a cell culture incubator, containing 10% CO2 for 30 min. Finally, the gels were detached from the sides of the well to enable the contraction of the matrix within 8 days. The media were changed daily.
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