MCM7 overexpressing stable cell lines were generated using the WT-MCM7 and Mut-MCM7 overexpression plasmids (OriGene Technologies). Plasmids were linearised using PsiI (New England Biolabs, Frankfurt am Main, Germany), and DNA band elusion was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) according to the user manual. N2a cells were plated on six-well plates and incubated 48 hours at 37°C. Cells were transfected overnight using Lipofectamine 2000 transfection reagent (Invitrogen) reduced serum medium (Opti-MEM GlutaMAX supplement, Gibco) and 2 µg of linearised overexpression plasmids without antibiotics. Trypsinisation was performed as previously described and cells were plated and incubated overnight at 37°C. Geneticin (600 µg/mL, G-418 Solution; Roche Diagnostics GmbH, Mannheim, Germany) was added to the cells, and the medium was changed every 48 hours for 7 days to obtain only cells with the integrated MCM7 overexpression plasmids.
Opti mem glutamax supplement
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Opti-MEM GlutaMAX supplement is a cell culture medium that provides essential nutrients for the growth and maintenance of cells in vitro. It contains a modified version of Opti-MEM I Reduced Serum Medium, which is supplemented with GlutaMAX, a stable L-glutamine alternative. The supplement is designed to support cell viability and proliferation in various cell culture applications.
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Lab products found in correlation
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
G418 solution, by Roche (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Neurobasal b27 medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using opti mem glutamax supplement
1
Generating Stable MCM7 Overexpressing Cell Lines
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MCM7 overexpressing stable cell lines were generated using the WT-MCM7 and Mut-MCM7 overexpression plasmids (OriGene Technologies). Plasmids were linearised using PsiI (New England Biolabs, Frankfurt am Main, Germany), and DNA band elusion was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) according to the user manual. N2a cells were plated on six-well plates and incubated 48 hours at 37°C. Cells were transfected overnight using Lipofectamine 2000 transfection reagent (Invitrogen) reduced serum medium (Opti-MEM GlutaMAX supplement, Gibco) and 2 µg of linearised overexpression plasmids without antibiotics. Trypsinisation was performed as previously described and cells were plated and incubated overnight at 37°C. Geneticin (600 µg/mL, G-418 Solution; Roche Diagnostics GmbH, Mannheim, Germany) was added to the cells, and the medium was changed every 48 hours for 7 days to obtain only cells with the integrated MCM7 overexpression plasmids.
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MCM7 overexpressing stable cell lines were generated using the WT-MCM7 and Mut-MCM7 overexpression plasmids (OriGene Technologies). Plasmids were linearised using PsiI (New England Biolabs, Frankfurt am Main, Germany), and DNA band elusion was performed using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) according to the user manual. N2a cells were plated on six-well plates and incubated 48 hours at 37°C. Cells were transfected overnight using Lipofectamine 2000 transfection reagent (Invitrogen) reduced serum medium (Opti-MEM GlutaMAX supplement, Gibco) and 2 µg of linearised overexpression plasmids without antibiotics. Trypsinisation was performed as previously described and cells were plated and incubated overnight at 37°C. Geneticin (600 µg/mL, G-418 Solution; Roche Diagnostics GmbH, Mannheim, Germany) was added to the cells, and the medium was changed every 48 hours for 7 days to obtain only cells with the integrated MCM7 overexpression plasmids.
2
Measles Virus Infection Assay
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Infection was performed at indicated multiplicities of infection (MOI) at day 2. MOCK-treated cells (no virus, infection medium only) served as control. Cells were washed once with PBS and then treated with a distinct MOI of MeV-GFP diluted in 250 µL Opti-MEM (Opti-MEM + GlutaMAX Supplement, Gibco, Paisley, Scotland, UK). At 3 h post-infection (hpi), the inoculum was removed and changed to standard or starvation medium.
3
Cell Culture and Transfection Protocols
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HEK293 (CRL-1573; ATCC) and HeLa (CCL-2; ATCC) cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) or FUGENE6 (Roche) according to the manufacturer’s protocol. HEK293 cell lines stably expressing HA-ephrin-B1 or EphB2 and membrane targeted myristoylated GFP (EphB2 [GFP]) have been described previously (Jørgensen et al., 2009 (link)). SK-N-BE(2) cells (human neuroblastoma, CRL-2271; ATCC) were cultured in reduced serum medium (Opti-MEM, GlutaMAX Supplement; Gibco) containing 10% FBS (Gibco).
U251 cells (09063001-1VL; Sigma-Aldrich) were cultured in MEM (Gibco) containing 10% FBS (Gibco), 2 mM glutamine, 1% nonessential amino acids, and 1 mM sodium pyruvate. All cell lines tested negative for mycoplasma contamination.
Primary forebrain neurons and cortical neurons were dissected from E15.5 mouse embryos, plated onto cell culture dishes coated with 1 mg/ml poly-d -lysine (Sigma-Aldrich) and 5 µg/ml laminin (Thermo Fisher Scientific), and cultured in Neurobasal-B27 medium (Gibco; Lauterbach and Klein, 2006 (link)).
U251 cells (09063001-1VL; Sigma-Aldrich) were cultured in MEM (Gibco) containing 10% FBS (Gibco), 2 mM glutamine, 1% nonessential amino acids, and 1 mM sodium pyruvate. All cell lines tested negative for mycoplasma contamination.
Primary forebrain neurons and cortical neurons were dissected from E15.5 mouse embryos, plated onto cell culture dishes coated with 1 mg/ml poly-
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