MCF10A cells were cultured in a 50:50 mix of Dulbecco's modified Eagle's medium/Ham's F12 medium (Sigma, Dublin, Ireland) supplemented with 5% horse serum, 1 μg/ml insulin, 20 ng/ml epidermal growth factor (EGF), 100 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone, and 2 mM l -glutamine. MCF10A cell lines stably expressing short hairpin RNA (shRNA) were generated by transfection with pSUPER vectors encoding shRNA targeting PDLIM2 (shPDLIM2: ACATAATCGTGGCCATCAA) or a control shRNA (shScramble: TGACATGATAATACTCTCT), as described previously [7] (link), using Lipofectamine 2000, following the manufacturer's protocol. Cells were cultured in the presence of 1 mg/ml G418 (Calbiochem, La Jolla, CA) for 4 to 6 weeks, at which time individual clones were screened for expression of PDLIM2 by Western blot analysis and shScramble clones and shPDLIM2 clones with > 60% suppression were selectively expanded for experiments.
Dulbecco s modified eagle s medium ham s f12 medium
Manufactured by Merck Group
Sourced in Ireland
Dulbecco's modified Eagle's medium/Ham's F12 medium is a cell culture medium formulation widely used for the in vitro cultivation of various cell types. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required to support cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
2 mercaptoethanol, by Fujifilm (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
L glutamine penicillin streptomycin solution, by Merck Group (1 mentions)
Gsk3 inhibitor chir99021, by Bio-Techne (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
2 protocols using dulbecco s modified eagle s medium ham s f12 medium
1
Generating PDLIM2 Knockdown MCF10A Cells
2
Generation of Rhesus Macaque iPSCs
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Rh-iPSCs were generated from Rh PBMCs. PBMCs were stimulated by anti-CD2/3/28-coated beads (Miltenyi Biotec, catalog no. 130-092-919). After 4 days, the PBMCs were transduced with SeV vectors harboring OCT3/4, KLF2, SOX2, c-MYC,30 (link) and SV40 large T antigen, and then seeded onto inactivated mouse embryonic feeder cells (MEFs). The cultured medium was gradually replaced with Rh-iPSC medium (Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium [Sigma] supplemented with 20% KO serum replacer [Thermo Fischer Scientific], 1% l -glutamine-penicillin-streptomycin solution [Sigma], 1% nonessential amino acids [Thermo Fischer Scientific], 10 mM 2-mercaptoethanol, and 5 ng/mL bFGF [Wako]), in addition to 3 μM GSK-3 inhibitor CHIR 99021 (Tocris) and 2 μM MEK1/2 inhibitor PD0325901 (Wako). The established iPSC clones were transfected with small interfering RNA L52730 (link) using Lipofectamine RNAi Max (Invitrogen) to remove the SeV vectors from the cytoplasm.
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