Primary goat myoblasts were isolated from semitendinosus tissues of the lamb goat, and were purified and cultured according to previously described protocol [42 (link)]. Briefly, cells were cultured in growth medium containing DMEM/F12 basic medium (Gibco, Grand Island, NY, USA), 20% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), and antibiotics (100 U/mL penicillin and streptomycin) (15140-122, Invitrogen Corp, Waltham, MA, USA) in 5% CO2 at 37 °C. For myoblast differentiation, we used differentiation media: the media were switched to a differentiation medium containing DMEM/F12 basic medium and 2% horse serum (Gibco, Grand Island, NY, USA). Additionally, HEK293T cells were grown in DMEM (Gibco, Grand Island, NY, USA) with 10% FBS. Culture medium was changed every day.
Dmem f12 basic medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
DMEM/F12 is a basic culture medium used for the in vitro cultivation of mammalian cells. It is a widely used formulation that provides essential nutrients, vitamins, and salts to support cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Relesr, by STEMCELL (2 mentions)
Human fgf2, by Thermo Fisher Scientific (2 mentions)
Mitomycin c, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Mem neaa, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Matrigel hesc qualified matrix, by Corning (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Hrmecs, by Procell (1 mentions)
Ribofect cp transfection kit, by RiboBio (1 mentions)
6 well plate, by Corning (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
A83 01, by Merck Group (1 mentions)
L ascorbic acid, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
10 protocols using dmem f12 basic medium
1
Isolation and Differentiation of Goat Myoblasts
2
Chickens Challenged with Virus
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Twenty 3-days-old and 20 10-days-old SPF leghorn chickens (Guangdong Dahuanong Animal Health Products Co., Ltd., China) were raised in separated negative-pressure isolators and randomly divided into two groups, respectively. Chickens in the challenge groups were inoculated intravenously with dose of 0.2 ml (1.0 × 107 TCID50/ml) virus. The control groups were inoculated intravenously with an equal volume of DMEM/F12 basic medium (Gibco, Australia). All chickens were monitored daily for clinical signs. The anal swabs of all chickens were collected from 0 to 21 dpi. The weight of all chickens was recorded weekly. At 21 dpi, all chickens were humanely euthanized. The liver, heart, spleen, and kidney were collected. And the weight of spleen was recorded. The spleen indexes were calculated by the spleen (milligram, mg) / body weight (gram, g). For histopathological examinations, samples were fixed in 4% paraformaldehyde. The remaining samples were stored at−80°C. The animal experiment protocol used in this study was approved by and performed under the guidance of the Committee on the Ethics of Animal Experiments of Institute of Animal Health, Guangdong Academy of Agricultural Sciences Experimental Animal Welfare Ethics Committee on 8 November, 2021 (Approve ID: SPF2021027). All efforts were made to minimize animal suffering.
3
Modulation of Endothelial Cell Function by miR-92a-3p
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HRMECs were purchased from Procell Life Science &Technology (Wuhan, China), which were authenticated by short tandem repeat profiling and cultured in a DMEM/F-12 basic medium (Gibco, USA) with 10% FBS (Gibco, USA). HRMECs were transfected with 50 nM mimic-92a-3p or mimic-NC (RiboBio, Guangzhou, China) using a riboFECTTM CP Transfection Kit (RiboBio), according to the manufacturer's instructions. Briefly, HRMECs were transfected 24 hours after being plated in 6-well plates (Corning, NY, USA) with a mixture of synthetic miRNA oligomers and transfection reagent, and added to cells in penicillin-streptomycin-free medium. Cells were then used for endothelial functional assays (tube formation, transwell assay, and wound healing) after 48 hours of transfection. Detailed functional assays are available in Supplementary Materials and Methods .
4
Culturing Human Trophoblast Stem Cells
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The hTSCs were established from human blastocysts according to the published protocol.16 hTSCs were cultured on Collagen IV‐coated plates and maintained in TS medium containing DMEM/F12 basic medium (Gibco), 0.1 mM 2‐mercaptoethanol (Sigma), 0.2% FBS, 0.5% Penicillin–Streptomycin, 0.3% HSA, 1% ITS‐X supplement (Sigma), 1.5 mg/mL L‐ascorbic acid, 50 ng/mL EGF (PeproTech), 2 mM CHIR99021 (Selleckchem), 0.5 mM A83‐01 (Sigma), 1 mM SB431542, 0.8 mM VPA and 5 mM Y27632 (Selleckchem). HUVECs were isolated from human umbilical vein. HUVECs were cultured on Collagen I‐coated plates and maintained in endothelial cell medium (ECM) (Sciencell).
5
Establishment and Culture of Mammary Epithelial Cells
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The mammary epithelial cell line, used in the current study, was established by Hu et al. using mammary tissue from Chinese Holstein dairy cow, and the details regarding establishment, and purification and chromosomal analysis of BMEC, were published previously [16 (link)]. The cells were cultured in 25 cm2 culture flasks (HyClone, South Logan, UT, USA) using Dulbecco’s modified Eagle medium/F12 (DMEM/F12) basic medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA) and 1× penicillin/streptomycin (HyClone) at 37 °C in 5% CO2. The cells grew to approximately 90% confluence and then were used for subsequent experiments.
6
Culturing Rat Hippocampal Neural Stem Cells
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Rat NSCs were obtained from hippocampi of neonatal rats and cultured in DMEM/F12 basic medium (Gibco), supplemented with 1% penicillin/streptomycin (Gibco), 20 ng/ml epidermal growth factor (EGF; Invitrogen, Carlsbad, CA, USA), 10 ng/ml basic fibroblast growth factor (bFGF; Invitrogen), and 1% B27 (Invitrogen) for 5-7 days. The medium was replaced at half volume with fresh medium every 3 days.
7
Human ESC Cultivation and Passaging
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The Ethics Committee of BGI-IRB approved this study. Human ESC lines H9 were cultured as previous description [57 (link)]. Briefly, cells were retrieved from liquid nitrogen tank and cultured in hESC medium (DMEM/F12 basic medium (Life Technologies), 20% knockout serum replacement (KSR, Life Technologies), 1 × L-glutamine (Life Technologies), 1 × MEM NEAA (Life Technologies), 0.1 mM 2-Mercaptoethanol (Life Technologies) and 50 ng/mL human FGF-2 (Life Technologies)) on mitomycin C (Sigma) treated murine embryonic fibroblasts (MEFs). To sustain undifferentiated states, cells were fed daily with fresh medium. For passaging, colonies were dispersed into small clumps with 1 mg/mL Collagenase IV (Life Technologies) for 20 min at 37℃, then plated onto Matrigel hESC-qualified Matrix (Corning)-coated dishes in mTeSR1 medium (Stemcell Technologies) at a ratio of 1:3 to 1:6. In the feeder-free medium, ReLeSR™ (Stemcell Technologies) were used for dissociation and passaging according to the manual.
8
Culturing Human Embryonic Stem Cells
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The Ethics Committee of BGI-IRB approved this study. H7 were obtained from GE healthcare and HUES1, HUES8, HUES9 lines were bought from Harvard University. All hESC lines with passage number between 30 and 40 were used in this study and cultured according to the protocol established in our lab. Briefly, cells were grown in hESC medium containing DMEM/F12 basic medium (Life Technologies), 20% knockout serum replacement (KSR, Life Technologies), 1×L-glutamine (Life Technologies), 1×MEM NEAA (Life Technologies), 0.1 mM 2-Mercaptoethanol (Life Technologies) and 50 ng/mL human FGF2 (Life Technologies) on Mitomycin C (Sigma) treated murine embryonic fibroblasts (MEFs), medium was changed every day. About 7 days, cells were dispersed into small clumps with 1 mg/mL Collagenase IV (Life Technologies) for 20 min at 37°C-, then plated onto Matrigel hESC-qualified Matrix (Corning)-coated dishes in mTeSRTM1 medium (Stemcell Technologies) at a ratio of 1:3 to 1:6. In the feeder-free medium, ReLeSRTM (Stemcell Technologies) were used for dissociation and passaging according to the manual.
9
Mouse Ovary Isolation and Culture
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The ovary isolation and culture were performed as reported by Cai et al. [49 (link)]. Briefly, the ovaries from 12.5, 13.5, 14.5, or 15.5 dpc mice were separated and washed by prechilled PBS three times, which were then cultured in 6-well culture dishes containing 1 ml of basic DMEM/F12 medium (Gibco, Life Technologies) at 37 °C in 5% CO2/95% air atmosphere with saturated humidity. Half of the medium was replaced every 2 days until the ovaries grew to the required stages. 25 μM D4476 was added to detect the effects of CK1α on folliculogenesis.
10
Ovarian Tissue Culture with Inhibitors
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Ovaries were separated by microdissection from the mesonephros or ovarian capsule in prechilled PBS (10 mM, pH 7.4) under a stereomicroscope (ZSA302, COIC). The isolated ovaries were cultured in 6‐well culture dishes (NEST Biotechnology) with basic DMEM/F‐12 medium (Gibco, Life Technologies) and Penicillin‐Streptomycin at 37°C in a 5% CO2, 95% air atmosphere with saturated humidity.
Ovaries were cultured in either medium with dimethylsulfoxide (DMSO) or medium supplemented with inhibitor. The concentration of inhibitors used in this study: GSK‐LSD1‐2Hcl (S7574; Selleck): 64 μM; 3MA (S2767; Selleck, USA): 2.5 mM; Z‐VAD‐fmk (S7023; Selleck): 50 μM; Chloroquine diphosphate (C6688; Sigma): 10 μM; Bafilomycin A1 (S1413; Selleck): 60 nM; MG132 (S2619; Selleck): 5 μM; Cycloheximide (HY‐12320; MedChemExpress): 2 nM; and SP2509 (S7680; Selleck): 5 nM.
Ovaries were cultured in either medium with dimethylsulfoxide (DMSO) or medium supplemented with inhibitor. The concentration of inhibitors used in this study: GSK‐LSD1‐2Hcl (S7574; Selleck): 64 μM; 3MA (S2767; Selleck, USA): 2.5 mM; Z‐VAD‐fmk (S7023; Selleck): 50 μM; Chloroquine diphosphate (C6688; Sigma): 10 μM; Bafilomycin A1 (S1413; Selleck): 60 nM; MG132 (S2619; Selleck): 5 μM; Cycloheximide (HY‐12320; MedChemExpress): 2 nM; and SP2509 (S7680; Selleck): 5 nM.
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