ES cells were dissociated in 0.25% trypsin-EDTA (Sigma-Aldrich) and reaggregated in 96-well low-cell-adhesion plates (Lipidure coat, NOF, Japan) at seeding densities of 3 × 103 and 3 × 104 cells per 150 µl of medium to induce cerebellar differentiation of mouse ES cells in serum-free culture of embryoid-body like aggregates (SFEBq)20 (link). The differentiation medium contained: Isocove’s modified Dulbecco’s medium (Gibco)/Ham’s F12 (Gibco) 1:1, chemically defined lipid concentrate (1% of total volume, Sigma-Aldrich), penicillin/streptomycin (Sigma-Aldrich), monothioglycerol (450 µM, Sigma-Aldrich), human apo-transferrin (15 µg/ml, Sigma-Aldrich), insulin (7 µg/ml, Sigma-Aldrich) and crystallization-purified BSA (5 mg/ml, Sigma-Aldrich). In addition, 4 × 106 ES cells28 (link) were seeded per Petri dish (Greiner Bio-one) in differentiation medium.
Isocove s modified dulbecco s medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Isocove's modified Dulbecco's medium is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It provides a defined and standardized nutrient composition to ensure consistent and reliable cell culture results.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (2 mentions)
Hydrocell 24 multi well plates, by Thermo Fisher Scientific (2 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Human apo transferrin, by Merck Group (1 mentions)
Monothioglycerol, by Merck Group (1 mentions)
Chemically defined lipid concentrate, by Merck Group (1 mentions)
Petri dish, by Greiner (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
5 protocols using isocove s modified dulbecco s medium
1
Cerebellar Differentiation of Mouse ES Cells
2
Cell Line Maintenance Protocol
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All lymphoma cell lines were maintained in Isocove’s Modified Dulbecco’s Medium (IMDM) (Gibco, USA) supplemented with 10% fetal bovine serum and 2 mM l -glutamine. 293T cells and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum.
3
Modulating Keloid Tissue Explants
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Keloid tissues were obtained from active-stage keloid patients (n = 4). Keloid tissue explants were prepared by dissecting keloid central dermal tissue into 2-mm diameter pieces with sterile 21-gauge needles. The explants were plated onto HydroCell® 24 multi-well plates (Nunc, Rochester, NY) after which they were cultured for 4 hr in Isocove’s modified Dulbecco’s medium (GIBCO) supplemented with 5% FBS, 10 mM insulin (Sigma) and 1 mM hydrocortisone (Sigma). For histological analysis, dEl-k35/LacZ or dEl-k35/sLRP6E1E2 at 1 × 1010 VP or 10 mM of commercially available Wnt inhibitor, JW 55 (Axon Medchem, Groningen, Netherland), were added into the plates containing keloid tissue explants, and incubated at 37 °C in 5% CO2 incubator for 3 or 5 days.
4
Keloid Spheroid Transduction with Adenoviral Vectors
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Keloid tissues were obtained from active-stage keloid patients (n = 5). Keloid spheroids were prepared by dissecting keloid central dermal tissue into 2-mm diameter pieces with sterile 21-gauge needles. Explants were plated onto HydroCell® 24 Multi-well plates (Nunc, Rochester, NY) after which they were cultured for 4 h in IMDM (Isocove’s modified Dulbecco’s medium, Gibco BRL) supplemented with 5% fetal bovine serum, 10 mM l−1 insulin and 1 mM l−1 hydrocortisone. Each of the Ads (dE1-RGD/GFP/shMot and dE1-RGD/GFP/scramble) at 1 × 1010 VP were added into the plates containing keloid spheroid, and incubated at 37 °C in 5% CO2 incubator for 3 days. The transduced keloid spheroids were then fixed with 10% formalin, paraffin-embedded, and cut into 5-μm-thick sections.
5
Evaluating Caudatin's Effects on HMC-1 Cells
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HMC-1 was maintained in standard conditions using Isocove’s modified Dulbecco’s medium (GIBCO, Grand Island, NY, USA) containing antibiotics (100 units/ml penicillin/100 μg/ml streptomycin) and 10% heat-inactivated fetal bovine serum (GIBCO) in a cell incubator (at 37 °C with 5% CO2). Cell was checked for mycoplasma contamination using Mycoplasma detection Kit (iNtRON Biotech, Sungnam, Korea). HMC-1 (3 × 105/well) was treated with caudatin (ChemFace, Wuhan, China) for 1 h before stimulation with PMACI. According to a prior study (Qiu et al., 2021 (link)), caudatin at concentrations of 0.5, 5, and 50 μM was used to study concentration-dependent effects. An evaluation of cell viability was attained by the MTT (Sigma Chemical Co., St. Louis, MO, USA) assay where a tetrazolium reagent is reduced to insoluble purple formazan crystals by mitochondria of viable cells.
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