The rat epithelial cell line C9 was purchased from American Type Culture Collection (Manassas, VA, USA). C9 cells were cultured in advanced Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, and seeded (7 × 103) on 96-well plates. After 24 h, the C9 cells were transfected with the miScript miRNA mimic (Qiagen; the final concentrations of miR-27a and miR-107 were 0.1 nM and 10 nM, respectively) using Lipofectamine 3000 reagent. Negative control cells were transfected with small RNAs and scrambled sequences. The medium was changed 24 h after transfection, and after another 24 h, the cells were collected for the TRIzol reagent.
Advanced dulbecco s modified eagle s medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Advanced Dulbecco's modified Eagle's medium is a cell culture medium formulation used to support the growth and maintenance of various cell types in laboratory settings. It provides the necessary nutrients and components to sustain cell viability and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Greiner (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Miscript mirna mimic, by Qiagen (1 mentions)
Geneporter 3000, by Genlantis (1 mentions)
Influx cell sorter, by BD (1 mentions)
Rock inhibitor, by STEMCELL (1 mentions)
Aggrewell 400 plate, by STEMCELL (1 mentions)
M csf, by R&D Systems (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Aggrewell medium, by STEMCELL (1 mentions)
Neomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Lonza (1 mentions)
6 ohda, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
11 protocols using advanced dulbecco s modified eagle s medium
1
miRNA Transfection in Rat C9 Cells
2
Stable Transfection of Amelanotic Melanoma
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Amelanotic tyrosinase-knockout B16-F10 melanoma cells were kindly provided by Dr Shweta Tikoo from the Centenary Institute, Sydney, Australia, and maintained in Advanced Dulbecco's modified Eagle's medium and 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, USA). They were stably transfected with CEFLP-tdTomato-H2B eukaryotic expression plasmids using GenePORTER ® 3000 (Genlantis, San Diego, CA, USA). Transfected cells were selected with puromycin (10 µg/mL) and tdTomato-expressing cells were purified by flow cytometry on a BD Influx Cell Sorter.
3
Characterization of Murine Prostate Cell Lines
4
Differentiation of Embryoid Bodies to iMacs
5
MDCK-SIAT2,6-UF Cells for Influenza Virus Isolation
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MDCK-SIAT2,6-UF cells [10 , 11 (link)], which overexpress influenza virus α2,6-linked sialic acid receptors, were used for the isolation of influenza viruses. The cells were propagated as monolayers at 37 °C and 5% CO2 in Advanced Dulbecco’s Modified Eagle’s Medium (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 2 mM L-Alanyl-L-Glutamine (GlutaMAX, Invitrogen Corp.), antibiotics (50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin (Invitrogen Corp.)), and 10% (v/v) low IgG, heat-inactivated gamma-irradiated fetal bovine serum (HyClone, Logan, Utah).
6
Vena Saphena Myofibroblast Cultivation
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Human vena saphena cells were harvested from the vena saphena magna obtained from patients according to Dutch guidelines of secondary use material and have previously been characterized as myofibroblasts (24 (link)). The myofibroblasts were cultured in advanced Dulbecco’s modified Eagle’s medium (Invitrogen, Breda, the Netherlands) supplemented with 10% fetal bovine serum (Greiner Bio-One, Monroe, NC), 1% penicillin/streptomycin (Lonza, Basel, Switzerland), and 1% GlutaMax (Invitrogen). Only cells with a passage lower than 7 were used in this study. To avoid cell-cell contact between the cells, the micropatterned substrates were seeded with a cell density of 500 cells cm−2 and cultured for 24 h at 37°C in 5% CO2.
7
Isolation and Culture of Vascular Myofibroblasts
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Human vascular-derived cells were harvested from segments of a vena saphena magna from a patient who underwent bypass surgery and were obtained according to the Dutch guidelines for secondary used materials. Cells were obtained using the outgrowth method and cultured using standard culture methods in a humidified atmosphere con-taining 5% CO 2 at 37°C as described previously. 8 (link) These cells were previously characterized as a-smooth-muscleactin positive and smoothelin and S100A4 negative, 7 indicating activated smooth muscle cells, which are also often referred to as myofibroblasts. Isolation and expansion medium consisted of advanced Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 1% GlutaMax (Gibco), 1% Penicillin/Streptomycin (P/S; Lonza), and 10% fetal bovine serum (FBS) (Greiner Bio-one).
8
Neuroblastoma SH-SY5Y Cell Culture Protocol
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The human neuroblastoma
SH-SY5Y cell line was purchased from American Type Culture Collection
(CRL-2266; VA, USA). These cells were cultured in Advanced Dulbecco’s
modified Eagle’s medium (Gibco, Thermo Fisher Scientific, MA,
USA) supplemented with 10% fetal bovine serum (Gibco), 2 mMl -glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (Sigma,
MO, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C and grown to 80% confluence. Prior to cell treatments,
the complete medium was replaced with serum-depleted medium. Compounds
were prepared as stock solutions of 20 mM in DMSO and were used at
concentrations of 5–50 μM. For the cytotoxic stimuli,
the 10 mM 6-OHDA (Sigma, MO, USA) stock was prepared in phosphate
buffered saline, pH 7.4, with 0.01% ascorbic acid. For the detailed
experimental procedures seeSupporting Information .
SH-SY5Y cell line was purchased from American Type Culture Collection
(CRL-2266; VA, USA). These cells were cultured in Advanced Dulbecco’s
modified Eagle’s medium (Gibco, Thermo Fisher Scientific, MA,
USA) supplemented with 10% fetal bovine serum (Gibco), 2 mM
MO, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C and grown to 80% confluence. Prior to cell treatments,
the complete medium was replaced with serum-depleted medium. Compounds
were prepared as stock solutions of 20 mM in DMSO and were used at
concentrations of 5–50 μM. For the cytotoxic stimuli,
the 10 mM 6-OHDA (Sigma, MO, USA) stock was prepared in phosphate
buffered saline, pH 7.4, with 0.01% ascorbic acid. For the detailed
experimental procedures see
9
HeLa and COS-7 Cell Cycle Synchronization
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HeLa and COS-7 cells from the American Type Culture Collection were maintained in advanced Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum (FBS; Hyclone) and penicillin–streptomycin (100 units/ml and 100 μg/ml, respectively; Gibco) at 37°C with 8% CO2.
For cell cycle synchronization, HeLa cells were first blocked in G1/S with 2.5 mM thymidine (Sigma) for 16 h and then released in fresh culture medium for 8 h to enrich mitotic cells. To inhibit Aurora B kinase activity, cells were treated with the Aurora B inhibitor ZM447439 (2458, TOCRIS) at 5 μM for 45 min after thymidine release.
For cell cycle synchronization, HeLa cells were first blocked in G1/S with 2.5 mM thymidine (Sigma) for 16 h and then released in fresh culture medium for 8 h to enrich mitotic cells. To inhibit Aurora B kinase activity, cells were treated with the Aurora B inhibitor ZM447439 (2458, TOCRIS) at 5 μM for 45 min after thymidine release.
10
Cell Line Maintenance and Cultivation
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The human cutaneous (c81‐61, C8161) and uveal (OCM‐1) melanoma cell lines have been described previously 2 , 27 and were maintained in RPMI‐1640 (Lonza, Breda, The Netherlands) supplemented with 10% fetal bovine serum gold (FBS; Fisher Scientific, Landsmeer, The Netherlands). The Ewing sarcoma cell lines EW7 and RDES 5 were maintained in RPMI‐1640 (Lonza) supplemented with 10% fetal bovine serum gold (FBS; Fisher Scientific) and 2 mm l ‐glutamine. Human perivascular cells from the vena saphena magna (HVSCs) were isolated in accordance to Dutch guidelines for the secondary use of material (https://www.federa.org ) and maintained in advanced Dulbecco's modified Eagle's medium (Gibco; Fisher Scientific) supplemented with 10% FBS, 2 mm glutamine, and 1% penicillin/streptomycin (Lonza). Human umbilical vein endothelial cells (HUVECs) and RF24 cells (EC‐RF24; immortalized HUVECs) were cultured as described previously 28 . All cells were cultured at 37 °C under a humidified atmosphere containing 5% CO2. Cell cultures were routinely confirmed to be negative for mycoplasma infection.
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