HUVEC-TERT2 (Evercyte GmbH, Vienna, Austria, CHT-006-0008) were cultured on a 0.1% gelatin (Merck KGaA, Burlington, MA, USA)-coated surface using endothelial cell growth basal medium (Lonza Group Ltd., Basel, Switzerland) supplemented with human endothelial growth factor, hydrocortisone, bovine brain extract (BBE), ascorbic acid, FBS from Lonza SingleQuots Kit (Lonza Group Ltd., Basel, Switzerland), and 20 µg/mL G418 solution (Roche Diagnostics, Basel, Switzerland). Cells were kept at 37 °C in a 5% CO2/95% air environment and subcultivated according to the vendor’s protocol, when cells reached 80% of confluence.
G418 solution
Manufactured by Roche
Sourced in Switzerland, Germany
G418 solution is a sterile-filtered antibiotic commonly used as a selective agent in cell culture and molecular biology applications. It contains the active ingredient geneticin, which inhibits the growth of eukaryotic cells not expressing a resistance gene.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Sh800 cell sorter, by Sony (2 mentions)
Endothelial cell growth basal medium, by Lonza (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Fugene 6, by Promega (1 mentions)
Opti mem glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Prostaglandin e1, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 luc2 tdt plasmid, by Addgene (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Gemini Bio (1 mentions)
Movas, by American Type Culture Collection (1 mentions)
15 protocols using g418 solution
1
HUVEC-TERT2 Cell Culture Protocol
2
Characterization of GHFT1 Cell Line
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GHFT1 cells (Dr Pamela Mellon, University of San Diego, La Jolla, CA) were maintained at 37°C/5% CO2 in Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (Hyclone, Logan, UT) and 100 units/ml penicillin-streptomycin (Invitrogen). All cultured cell lines employed in this study were authenticated by RNA-Seq analysis performed in the lab (Figure 8—source data 1 ). This authentication was done based on the positive expression of Pou1f1 and absence of expression of Prolactin, Growth hormome and Thyroid-Stimulating Hormone. Prop1 cDNA was cloned into pEF1α-FLBIO-puro (Kim et al., 2009 (link)) to be N-terminally tagged by standard cloning using the BamHI restriction site. The BirA expression vector used was pEF1α-BirAV5-neo (Kim et al., 2009 (link)). Both plasmids were a gift from Alan B. Cantor (Boston Children’s Hospital, Boston, MA). Cells were plated onto 24-well plastic plates (Fisher Scientific, Fair Lawn, NJ) at a density of 3.5 × 104 cells/well. Prop1 and BirA expressing vectors were transfected into cultured cells using the FuGENE 6 (Promega) at a 3:2 ratio according to the manufacturer’s protocol. To make stable cell lines, transfected cells were incubated with 1.4 mg/ml G-418 solution (Roche) and 5 µg/ml Puromycin (Sigma) for selection of BirA and Prop1-Tag, respectively.
3
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.
4
Overexpression of Tie2 in OSCC Cells
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OSCC-derived cells (SAS and Sa3) were transfected with Tie2 vector designed to cause overexpression of Tie2 cDNA (oeTie2 cells) (OriGene Technologies, Rockville, MD, USA) and non-targeting vector (Mock cells) (OriGene Technologies) using Lipofectamine 3000 (Invitrogen). After transfection, the cells that stably overexpressed Tie2 were isolated by the selection marker, G-418 Solution (Roche Diagnostics). Two to three weeks after incubation with G-418 Solution, individual clones were isolated and used for further experiments.
5
Culturing DRG Neuronal Cell Line HD10.6
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The human DRG neuronal cell line HD10.6 cell line was essentially described [18 (link),19 (link),20 ,21 (link)]. HD10.6 is cultured on a coating layer consists of 16.6 μg/mL fibronectin, and in advanced Dulbecco’s modified Eagle medium-nutrient mixture F-12 (DMEM/F12, Gibco, Waltham, MA, Cat#12634-010), supplemented with 1× B27 (Gibco™, Cat#: 17504044), 1× Glutamax, (Gibco™, #35050061), 10 ng/mL Prostaglandin E1 (Sigma-Aldrich, St. Louis, MO, Cat#: P5515), 0.5 ng/mL basic fibroblast growth factor (β-FGF), and 50 μg/mL G418 solution (Roche, Basel, Switzerland, Cat#: 04727878001).
6
Establishment of Dual-Labeled GL261 Glioma Cell Line
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GL261 glioma cells obtained from Prof. Helmut Kettenman (MDC, Berlin, Germany), were cultured in Dulbecco modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, MD, USA) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37 °C (Heraeus, Hanau, Germany).
GL261 luc+/tdT+ cell line was developed from the GL261 cells. GL261 cells were seeded in antibiotic-free medium and after 24 h transfected with pcDNA3.1(+)/Luc2-tdT plasmid (Addgene) linearized with Notl restriction enzyme (ThermoFisher) using Lipofectamine2000 (ThermoFisher). Cells were maintained in a medium supplemented with 400 µg/mL G-418 Solution (Roche) until complete death of mock-transfected cells. tdT-positive cells were enriched by fluorescence-activated cell sorting, expanded and cryopreserved in FBS with 10% dimethyl sulfoxide (Sigma-Aldrich).
GL261 luc+/tdT+ cell line was developed from the GL261 cells. GL261 cells were seeded in antibiotic-free medium and after 24 h transfected with pcDNA3.1(+)/Luc2-tdT plasmid (Addgene) linearized with Notl restriction enzyme (ThermoFisher) using Lipofectamine2000 (ThermoFisher). Cells were maintained in a medium supplemented with 400 µg/mL G-418 Solution (Roche) until complete death of mock-transfected cells. tdT-positive cells were enriched by fluorescence-activated cell sorting, expanded and cryopreserved in FBS with 10% dimethyl sulfoxide (Sigma-Aldrich).
7
Anti-HBV and Anti-HIV Drug Susceptibility Assays
8
Proteasomal degradation of cODC
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The degron sequence of ODC is recognized directly by proteasomes, which leads to immediate destruction of the involved protein. The retroviral expression vector pQCXIN‐ZsGreen‐cODC, containing green fluorescence Gdeg, was kindly provided by Dr Frank Pajonk (Jonsson Comprehensive Cancer Center, UCLA, CA, USA) to S. Tanaka. The vector was transfected into platinum retroviral packaging cells, and the retrovirus collected from the supernatant was used to infect KM12SM cells. Stable transfectants were selected with G418 solution (Roche). The top 0.86% of cell populations of the EGFP channel were enriched by sorting three times using flow cytometry (Cell Sorter SH800; SONY) and maintained in 0.1 mg/mL G418 solution.
9
Modulation of Vascular Smooth Muscle Cell Inflammation
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Experiments were performed using the mouse aortic VSMC line MOVAS (ATCC® CRL-2797™, Manassas, VA, United States). Cells were maintained in High Glucose Dulbecco’s Modified Eagle Medium [High Glucose-DMEM (Sigma Chemical Co., St. Louis, MO, United States)] supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.2 mg/mL of the G-418 solution (Roche Diagnostics, Manheim, Germany). VSMC were incubated at 37°C in a humidified 5% CO2 atmosphere. Cells from passages 3-12 were used for the experiments. Cells were starved in High Glucose-DMEM, 0% FBS and 0.2 mg/mL of the G-418 solution. Afterwards, cells were stimulated with 10 ng/mL IL-1β for 1 h with or without pretreatment for 24 h with the Nrf2 activator tBHQ (20 μM) or for 1 h with apocynin (30 μM), catalase (1,000 U/mL) or the general intracellular TLR4 inhibitor CLI-095 (1 μM). In other set of experiments, cells were stimulated with hydrogen peroxide (H2O2, 100 μM, 1 h) or lipopolysaccharide (LPS, 10 μg/mL, 3 h) in the absence or the presence of tBHQ (20 μM, 24 h).
10
Establishing CEACAM1 Overexpression and Knockdown Cell Lines
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Transfection and RNA interference techniques were previously described7 (link)–9 (link),15 (link),20 (link). CEACAM1-4L or -4S cDNA was cloned into the pH-β actin vector23 (link), and was mixed with 1 μL of Lipofectamine 3000 (Invitrogen) to a final volume of 100 μL of Opti-MEM medium, and was added to NUGC3 cells grown to 80% confluence in 24-well plates. Forty-eight hours after transfection, a G418 solution (Roche, Basel, Switzerland) was added. The stably transfected cells were maintained in the culture media described above. Short hairpin RNA (shRNA) plasmids designed to target CEACAM1 were synthesized by SABiosciences (Frederick, MD, USA), as described before7 (link). Each plasmid (0.8 μg) was transfected into MKN7 cells as described above. The expression level of CEACAM1 of the stably transfected cell lines was evaluated by reverse transcription-polymerase chain reaction (Fig. 5A )
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