Human proximal renal tubular epithelial cells (HK-2) were purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China) and cultured in DME/F12 medium (Gibco, United States) supplemented with 1% penicillin-streptomycin (Gibco, United States) and 10% fetal bovine serum (FBS, Gibco, United States) under 5% CO2 and 95% air atmosphere at 37°C. HK-2 cells were cultured in a medium containing CoCl2 (7791-13-1, Changsha Jingkang New Material Technology Co. LTD., China) without nutrients (serum-free, glucose-free) for 6 h. The medium was then replaced with DME/F12 with or without mefunidone for 12 h to induce the hypoxia/reoxygenation (H/R) model.
Dme f12 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
DME/F12 medium is a cell culture medium formulated for the growth and maintenance of a variety of cell types. It is a combination of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture. This medium provides a balanced set of nutrients, vitamins, and other components required for cell proliferation and survival in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
Syringe filter, by Merck Group (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Egf 1, by Thermo Fisher Scientific (1 mentions)
Heat inactivated horse serum, by Thermo Fisher Scientific (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Lapatinib, by LC Laboratories (1 mentions)
Pa1 511a, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Merck Group (1 mentions)
Mir 20b mimic, by RiboBio (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
23 protocols using dme f12 medium
1
Hypoxia-Reoxygenation Model in HK-2 Cells
2
Clear Cell Renal Cell Carcinoma Study
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This study enrolled subjects visiting the Department of Urology, Shanghai Ruijin Hospital between September 2018 and August 2021. Their clear cell renal cell carcinoma (ccRCC) tissues and adjacent normal tissues (ANT) were obtained during the surgical operation. The study conformed to the standards set by the Declaration of Helsinki was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, China (approval number: 2018‐381). All subjects gave written informed consent before their participation. Twenty‐eight pairs of samples including both ccRCC and ANT tissues were collected after surgery for the following experiments. ccRCC cell lines 786‐O and 769‐P and human proximal tubular epithelial cell line HK‐2 were provided by the National Collection of Authenticated Cell Cultures. 786‐O cells and 769‐P cells were immediately cultured in RPMI1640 medium (Gibco, Waltham, MA, USA) and HK‐2 cells were cultured in DME/F12 medium (Gibco) when the cell lines were obtained. All the culture media contained 10% fetal bovine serum (FBS), 100 U·mL−1 penicillin, and 100 μg·mL−1 streptomycin, and the cultured cells were kept in 95% humidity and 37 °C 5% CO2 incubator (Thermo, Waltham, MA, USA).
3
Isolation and Amplification of Fowl Adenovirus
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Liver tissue samples were homogenized in phosphate-buffered saline (PBS ), followed by filtration through a Millipore syringe filter with a pore size of 0.22 μm to produce an inoculum for viral isolation after undergoing 3 freeze-thaw cycles. Afterwards, the resulting supernatant was used to infect LMH cells grown as monolayer in DME/F-12 medium (Gibco) supplemented with 10% fetal bovine serum at 37°C under humidified conditions containing 5% CO2. The cells were observed daily to monitor any cytopathic effects. When 80% cytopathies were observed, both the supernatant and infected cells underwent 3 additional freeze-thaw cycles prior to collection. After 3 round of plaque purification procedure and exogenous pathogenic microorganism detection, the FAdV-positive supernatants were subsequently amplified on confluent monolayers of LMH cell. The purified strains were identified using PCR to rule out other viruses such as AIV, IBV, IBDV, NDV, FAdV-4, and CIAV.
4
Isolation and Culture of Bovine Mammary Epithelial Cells
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Firstly, fresh udder tissues were washed several times until the solution was pellucid and without milk. Secondly, the tissues were cut about 1 mm3 and were washed again with PBS. The smaller tissues were cultured in DME/F-12 medium (Gibco, Waltham, MA, USA) with 10% fetal calf serum (Gibco, Waltham, MA, USA), 5 μg/mL insulin, 100 U/mL penicillin and streptomycin (Gibco, Waltham, MA, USA), 10 ng/mL epidermal growth factor 1 (EGF-1, Gibco, Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO2. After about 1 week, the purified GMECs were received.
5
MCF10A Cell Culture and Drug Treatment
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MCF10A cells were grown as described20 (link) in DME:F12 medium (Gibco BRL, Grand Island, NY) supplemented with 10 μg ml−1 insulin, 0.1 μg ml−1 cholera toxin, 0.5 μg ml−1 HC, 5% heat-inactivated horse serum (Gibco BRL) and 10 ng ml−1 EGF. The cells were starved overnight in medium, and thereafter stimulated with EGF (10 ng ml−1) or DEX (100 nM). siRNA transfections used Oligofectamine (Invitrogen) and ON-Target SMART (Dharmacon, Lafayette, CO) oligonucleotides. Lapatinib (di-p-Toluenesulfonate salt) was purchased from LC Laboratories and formulated as a suspension in 0.5% hydroxypropyl methylcellulose and 0.1% Tween 80. The drug was administered via oral gavage as a single bolus dose. Mig6-specific short hairpin RNA particles were produced in 293FT cells using the corresponding plasmid.
6
Characterization of MCF10A Breast Cell Line
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Unless indicated, cells were obtained from the American Type Tissue Culture Collection (ATCC). MCF10A cells were cultured in DME:F12 medium (Gibco BRL, Grand Island, NY, USA) supplemented with insulin (10 μg/ml), cholera toxin (0.1 μg/ml), hydrocortisone (0.5 μg/ml), heat-inactivated horse serum (5%; Gibco BRL) and EGF (10 ng/ml). The following antibodies were used: anti-DUSP1 (clone EPR18884 from Abcam), polyclonal anti-EGR1 (C-19; Santa Cruz), anti-FOS antibody (H-125; Santa Cruz), an antibody specific to the K27 acetylated form of histone 3 (from Abcam), a rabbit anti-GR polyclonal antibody (PA1-511A; Thermo Fisher Scientific) and an antibody to the N-terminus of RNA polymerase II (from Cell Signaling). Protein content was estimated using the BCA kit (from Sigma) and HBEGF was assayed using an ELISA kit (DuoSet, from R&D Systems, Minneapolis, MN, USA).
7
Breast Cancer Cell Line Culture and miR-20b Modulation
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Human normal mammary epithelial cell line MCF-10A was cultured in DME/F12 medium (Gibco, USA) containing 5% horse serum, 10 μ g/ml insulin, 20 ng/ml EGF, 100 ng/ml choleratoxin, 0.5 μg/ml hydrocortisone, 100 IU/ml penicillin and 100 μg/ml streptomycin. Human breast cancer cell lines MCF-7 and T-47D were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/ml penicillin and 100 μg/ml streptomycin. Other human breast cancer cell lines ZR-75-30 and SK-BR-3 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin in humidified 5% CO2 at 37°C. MiR-20b mimics, miR-20b inhibitor, antagomir-20b and their negative control were synthesized by Ribobio (RiboBio Co. Ltd., Guangzhou, China) [29 (link)].
The miR-20b gain-of-function study was performed using MiR-20b mimics (50 nM) and its negative control (50 nM) on the breast cancer cell lines. The loss-of-function study was performed with miR-20b inhibitor (100 nM) and its negative control (100 nM) on the cell lines. The cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions.
The miR-20b gain-of-function study was performed using MiR-20b mimics (50 nM) and its negative control (50 nM) on the breast cancer cell lines. The loss-of-function study was performed with miR-20b inhibitor (100 nM) and its negative control (100 nM) on the cell lines. The cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions.
8
Tumorsphere Formation Assay for Breast Cancer
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This assay tests the ability of single cells to form tumorspheres, the in vitro surrogate of stem-like cell. Breast tumor cells (2 × 105 cells/well in 1.5 ml of medium) were distributed into ultralow attachment 6-well plates. Tumorspheres were grown in DME-F12 medium (Gibco), supplemented with penicillin/streptomycin, L-glutamine and B27 supplement (1x, #17504–044, Life technologies), fibroblast growth factor-2 (FGF-2) (20 ng/ml, #13256029, Gibco) and hEGF (20 ng/ml, #10605-HNAE, Gibco), and allowed to grow for 7 to 10 days, or until the majority of spheres reached a diameter of 60 μm. Tumorspheres were counted and then harvested for protein extraction or splitted for second generation of tumorspheres and next lysed for protein extraction [12 (link)].
9
MCF10A Cell Culture and RNA Metabolic Labeling
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MCF10A cells were cultured as described (32 (link)) in DME:F12 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10 μg ml−1 insulin, 0.1 μg ml−1 cholera toxin, 0.5 μg ml−1 hydrocortisone, 5% heat-inactivated horse serum (Biological Industries, Beit-Haemek, Israel) and 10 ng ml−1 EGF. For time course experiments, cells were starved overnight in medium without additives, and thereafter stimulated with EGF (10 ng ml−1) or dexamethasone (DEX; 100 nM). RNA metabolic labeling was performed in different concentrations of 4-thiouridine (4sU; Sigma), as recommended before (35 (link)). The labeling reagent was added to the medium (0.5 mM final concentration) and incubated with cells for 20 min. This was followed by 40 min of labeling (0.3 mM) and further incubations for 1, 2 or 4 h (0.2 mM). When treated with EGF, cells were concomitantly labeled with 4sU.
10
Porcine Trophectoderm Cell Line Response to GABA and H2O2
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The porcine trophectoderm cell line-2 (pTr-2) was kindly provided by Prof. Zhenlong Wu (China Agricultural University, Beijing, China). pTr-2 cells were cultured in DME/F12 medium (Gibco, New York, NY, USA) containing 10% (v/v) fetal bovine serum (Gibco, New York, NY, USA) and 0.1% (v/v) insulin (5 mg/mL, Solarbio, Beijing, China) at 37 °C and 5% CO2. Cells were treated with GABA (0, 25, 50, 100, or 200 μmol/L) for 24 h prior to stimulation with 0.6 mmol/L H2O2 for 2 h.
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