The stable re-expression of LA and LC in the Lmna-KO MEFs was achieved through transfecting with PB533-based piggyBac transposon system vectors and the mouse codon-optimized piggyBac transposase (mPBase) expression vector, based on a previous report (Kirschner et al., 2015 (link)). Briefly, 0.4 µg pcDNA3-mPB (Cadiñanos and Bradley, 2007 (link)) with 1.6 μg PB533-mEmerald-LA-Neo and/or PB533-mEmerald-LC-Neo onto 35 mm dishes, respectively, using Lipofectamine 3000 by reverse transfection method, described above. PB533-mEmerald-C1-Neo was used as control. Transfected cells were selected by incubation in medium containing 400 µg/ml G-418 disulfate aqueous solution (Nacalai Tesque) for 7 d. pcDNA3-mPB was provided by Masashi Narita (Cancer Research UK Cambridge Institute, University of Cambridge, UK).
G418 disulfate aqueous solution
Manufactured by Nacalai Tesque
Sourced in Japan
G418 disulfate aqueous solution is a laboratory reagent used for the selection and maintenance of mammalian cell lines containing the neomycin resistance gene (neo). It is a broad-spectrum antibiotic that inhibits protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Nacalai Tesque (2 mentions)
Penicillin, by Nacalai Tesque (2 mentions)
Six well plate, by Thermo Fisher Scientific (1 mentions)
Sh800, by Sony (1 mentions)
Pegfp c1 flag ku80, by Addgene (1 mentions)
Pb533a 2, by System Biosciences (1 mentions)
Dmem high glucose, by Fujifilm (1 mentions)
Rpmi 1640 medium, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
4d nucleofector, by Lonza (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Pcmv6 entry mock, by OriGene (1 mentions)
Amaxa p3 primary cell 4d nucleofector x kit, by Lonza (1 mentions)
Nucleofector 2b, by Lonza (1 mentions)
Amaxa human stem cell nucleofector kit 2, by Lonza (1 mentions)
10 protocols using g418 disulfate aqueous solution
1
Stable Lamin Expression in Lmna-KO MEFs
2
Culturing and Differentiating Human Intestinal Epithelial Cells
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The African green monkey kidney cell line, Vero cells (ATCC®CCL-81™), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Vero cells expressing TMPRSS2, VeroE6/TMPRSS2 cells (VeroE6/TMPRSS2, JCRB number; JCRB1819), were maintained in DMEM containing 5% FBS and 1 mg/mL G418 disulfate aqueous solution (Nacalai Tesque) at 37 ℃. IEC#17 cells were differentiated from a human iPSC line, TkDN4-M, as described previously33 (link). IEC#20, IEC#25, and IEC#29 were also differentiated from the human iPSC lines, 1231A3, 1383D636 (link), and TkPP737 (link), respectively. To prepare monolayers, IECs were cultured in a multiwell plate at a density of 6.3 × 105/cm2 in a culture medium for 2 days. For differentiation, cells were cultured in a differentiation medium for 4 days (for details, refer to Ref.38 (link)). After 6 days, an average of 1 × 105 differentiated IECs were seeded in 96-well plates.
3
Stable Cell Lines for Fluorescent Fusion Proteins
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Cells were plated in a six-well plate (Thermo Fisher Scientific) 1 day before the transfection of the expression constructs for mCherry-tagged proliferating-cell nuclear antigen (PCNA; Leonhardt et al., 2000 (link)) or EGFP-ATM based on PB533A-2 (System Biosciences) with a PiggyBac transposon expression vector (PB210PA-1; System Biosciences), or pEGFP-C1-FLAG-Ku80 (Addgene 46958 ; Britton et al., 2013 (link)) using FuGENE HD Transfection Reagent (Promega) according to the manufacturer's instruction. The EGFP-ATM expression vector was constructed using a plasmid containing ATM cDNA provided by Tsuyoshi Ikura (Kyoto University, Japan) and the entire ATM sequence was verified by sequencing. To obtain a stable cell line, 2 days after the transfection, the cells were incubated in the presence of 1 mg/ml G418 disulfate aqueous solution (Nacalai Tesque) in the complete medium for >1 week. Cells that exhibited mCherry–PCNA or EGFP–ATM fluorescence were sorted using a cell sorter (Sony; SH800) and cultured in fresh medium without G418.
4
Establishment and Maintenance of Gastric Cancer and HEK293A Cells
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85As2 cells were established from MKN-45 human gastric cancer cells as described previously [11 (link)]. Cells were maintained in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate under a 5% CO2 atmosphere at 37°C. Human embryonic kidney (HEK) 293A cells stably expressing GHS-R were maintained in Dulbecco’s modified Eagle’s medium (DMEM; high-glucose; Wako, Osaka, Japan) supplemented with 10% FBS, 100 IU/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, and 500 μg/mL G418 Disulfate Aqueous Solution (Nacalai Tesque, Inc.) under a 5% CO2 atmosphere at 37°C.
5
Stable Transfection of IL13RA2 in HaCaT Cells
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Plasmids pCMV6-Entry (Mock) and IL13RA2 (Myc-DDK-tagged), which contains a cytomegalovirus promoter, and the IL13RA2 (NM_000640) human cDNA open reading frame clone were obtained from Origene Technologies (Rockville, MD, USA). The plasmids (1 µg) were dissolved in Amaxa P3 Primary Cell 4D-Nucleofector X Kit and were transfected into HaCaT cells using 4D-Nucleofector (Lonza, Basel, Switzerland), according to the manufacturer’s protocol. Transfected cells were then selected in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich, St. Louis, MO, USA) with 5% fetal bovine serum, Modified Eagle’s Medium Non-Essential Amino Acids, 10 mM HEPES, 1 mM sodium pyruvate (Thermo Fisher Scientific), and G418 disulfate aqueous solution (1500 µg/mL, Nacalai Tesque, Kyoto, Japan) for 3 weeks to obtain a stable cell line.
6
Generation of Myogenic MELAS-iPSCs
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We introduced a myogenic differentiation system34 (link) into MELAS-iPSCs using a piggyBac vector, PB200_hMyoD,35 (link) including (Tet-On)-MYOD-IRES-EGFP cDNA and a neomycin resistance gene. A01 MELAS-iPSCs were transfected with a transposase-expressing plasmid, pHL-EF1a-hcPBase34 (link) (1.5 μg), and PB200-hMyoD (1.5 μg) using Nucleofector 2b (Lonza) and an Amaxa human stem cell Nucleofector kit 2 (Lonza), according to the manufacturer’s protocol, and were then plated on feeder cells. Forty-eight hours after the transfection, a G418 disulfate aqueous solution (50 μg/mL; Nacalai Tesque) was added to select appropriate MyoD-iPSC clones with high EGFP expression.
7
Measuring GPCR Activation via cAMP
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GloSensor cyclic adenosine monophosphate (cAMP) assay (Promega, Madison, WI, USA) was used to measure G protein activation according to the manufacturer’s protocol. Briefly, HEK293-MOR-FLAG cells that stably expressed μ-opioid receptors were plated at 1.5 × 104 cells/µl in 96-well plates in high-glucose Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 700 µg/ml G418 disulfate aqueous solution (Nacalai Tesque, Kyoto, Japan). Next day, cells were transiently transfected with GloSensor cAMP Plasmid (Promega) using the FuGENE HD Transfection Reagent (Promega). Twenty-four hours after transfection, medium is changed to equilibration medium (88% CO2-independent medium, 10% FBS, 2% GloSensor cAMP Reagent stock solution), and then cells were incubated for 2 h. After 5-min application with forskolin (130 µM), drugs were treated to the well for 30 min. Chemiluminescence in each step was measured by a microplate reader (GLOMAX, Promega).
8
Establishment and Maintenance of PC12-VG Cells
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PC12-VG cells were previously established by the stable transfection of the β-galactosidase reporter gene transcribed from the vasoactive intestinal peptide (VIP) gene promoter into the parental rat pheochromocytoma PC12 cell line
[13 (link)]. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH), 5% horse serum (Gibco, Life Technologies, Carlsbad, CA), penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (nacalai tesque, Kyoto, Japan), and 100 μg/ml G418 disulfate aqueous solution (nacalai tesque), as previously described
[13 (link)].
[13 (link)]. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH), 5% horse serum (Gibco, Life Technologies, Carlsbad, CA), penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (nacalai tesque, Kyoto, Japan), and 100 μg/ml G418 disulfate aqueous solution (nacalai tesque), as previously described
[13 (link)].
9
Derivation of Chimeric Embryos from ES Cells
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Female ICR mice were treated with pregnant mare serum gonadotropin (PMSG; 7.5 IU) and human chorionic gonadotropin (hCG; 7.5 IU) by intraperitoneal injection. Embryos were rinsed with M2 medium (Sigma) and cultured in KSOM medium until development into blastocysts. WT, Dnmt3a KO, and Dnmt3b KO 2i/L ES cells were trypsinized, and 7–8 cells per embryo were injected into the blastocoels of E3.5 blastocysts. Injected blastocysts were transferred into the uteri of pseudopregnant ICR mice. A Fully Automated Fluorescence Stereo Microscope Leica M205 FA (Leica MICROSYSTEMS) was used to observe GFP-labeled embryos. MEFs were isolated from E14.5 embryos and cultured in DMEM (Nacalai Tesque) with 2 mM L-glutamine (Nacalai Tesque), 100× nonessential amino acids (NEAA) (Nacalai Tesque), 100 U/ml penicillin, 100 μg/ml streptomycin (P/S) (Nacalai Tesque), and 10% fetal bovine serum (FBS) (GIBCO). To remove host cells, GFP-labeled MEFs were collected by fluorescence activated cell sorting (Aria II, BD) after selection with 350 μg/ml G418 disulfate aqueous solution (Nacalai Tesque) for 7 days.
10
Establishing Dox-inducible Cell Lines for Experimentation
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Human osteosarcoma U2OS, human embryonic kidney (HEK) 293T, and human cervical cancer HeLa S3 cells, purchased from the American Type Culture Collection, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, D5796) supplemented with 10% fetal bovine serum (FBS) (HyClone, SV30014.03) with 100 U/mL penicillin and 100 μg/mL streptomycin (Nacalai Tesque, #26253-84). For preparation of Dox-inducible U2OS advanced cells, pTet-On advanced plasmid vector (Clontech) was transfected into cells. The cells were incubated in DMEM supplemented with 10% FBS, penicillin/streptomycin, and 0.2 mg/mL G418 disulfate aqueous solution (Nacalai Tesque, #16513-26), from which several single colonies were selected for preparation of Tet-ON inducible cell lines. For culture of pTet-On_advanced cells, 0.2 mg/mL G418 was added to the medium.
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