MEF cells were immortalized from PerkΔex7-9/Δex7-9 embryos55 (link) and PerkC528X/C528X embryos using a plasmid carrying the SV40 large T antigen (SV40 1: pBSSVD2005, Addgene, plasmid #21826, deposited by David Ron). Following immortalization, MEF cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini, West Sacramento, CA, USA) and 1 × penicillin-streptomycin (Pen-Strep) at 100 U/mL–100 μg/mL (Gibco). Mouse MIN6 (Dr. Jun-Ichi Miyazaki, Osaka University, Japan) beta cells and human AD293 cells (Agilent, Santa Clara, CA, USA) were cultured under the same conditions as MEF cells. Primary human cadaveric islets were obtained from Prodo Labs of Integrated Islet Distribution Program (IIDP). Upon receipt, islets were transferred from shipping media to CMRL 1066 (Connaught Medical Research Laboratories, Toronto, ON, Canada; purchased from Gibco) supplemented with 10% FBS, 1× Pen-Strep, and 2 mM L-glutamine (Gibco) at a concentration of 800–1,000 islet equivalents (IEQ) per milliliter in a non-tissue-culture-treated 6 cm dish and cultured overnight. All cells were cultured in a humidified, 5% CO2 incubator at 37°C.
Sv40 1 pbssvd2005
Manufactured by Addgene
The SV40 1: pBSSVD2005 is a plasmid vector that contains the SV40 early promoter and enhancer sequences. This vector is commonly used for the expression of genes in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (2 mentions)
Human ad293 cells, by Agilent Technologies (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
2 protocols using sv40 1 pbssvd2005
1
Immortalization and Maintenance of MEF, MIN6, and AD293 Cells
2
Immortalization of Mouse Embryonic Fibroblasts
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Mouse embryonic fibroblasts (MEF) cells were immortalized from Perk Dex7-9/Dex7-9 52 and Perk C528X/C528X mice using a plasmid carrying the SV40 large T antigen (SV40 1: pBSSVD2005, Addgene, plasmid #21826, deposited by David Ron). Following immortalization, MEF cells were maintained in Dulbecco's Modified Eagle Medium, DMEM (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum, FBS (Gemini, West Sacramento, CA) and 1× Penicillin-Streptomycin (Pen-Strep) at 100U/ml-100μg/ml (Gibco). Mouse MIN6 (Dr. Jun-Ichi Miyazaki, Osaka University, Japan) beta cells and human AD293 cells (Agilent, Santa Clara, CA) were cultured under the same conditions as MEF cells. Primary human cadaveric islets were obtained from Prodo Labs of Integrated Islet Distribution Program (IIDP). Upon receipt, islets were transferred from shipping media to CMRL 1066 (Connaught Medical Research Laboratories, Toronto, Canada; purchased from Gibco) supplemented with 10% FBS, 1× Pen-Strep and 2mM L-Glutamine (Gibco) at a concentration of 800-1000 islet equivalents (IEQ) per milliliter in a non-tissue culture treated 6 cm dish and cultured overnight. All cells were cultured in a humidified, 5% CO2 incubator at 37°C.
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