Both the calcium phosphate precipitation method [29 ] and the lipofection method using the Lipofectamine 2,000 Reagent (Invitrogen, CA, USA) were used to transfer the Geneticin® resistant gene, pSV2neo, into the yellowtail fin cells. The treated cells were cultured in L-15 medium (Invitrogen, USA) containing Geneticin® (1 mg/mL, Invitrogen, USA) at 22°C to select the transformed cells. More than 300 independent Geneticin®-resistant clones were pooled for the fusion experiments. These cells were irradiated by exposure to 3,000, 4,000, 5,000, 6,000, 8,000, or 10,000 rads (MBR-1520R; Hitachi Power Solutions Co., Ltd., Ibaraki, Japan), and were fused with derivative mouse B78 cells at a 2:1 ratio in the presence of polyethylene glycol 1500 (Roche, Basel, Switzerland). The cells were cultivated with DMEM medium (Invitrogen, USA) containing Geneticin® for 3–4 weeks until hybrid clone colonies appeared. DNA was extracted from individual clones using a DNAdvance Kit (Beckman Coulter, CA, USA).
Geneticin
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, Germany, United Kingdom, Canada, Belgium, Australia, Japan, Switzerland, Spain, Italy, Sweden, Thailand
Geneticin is a broad-spectrum antibiotic used for the selection of mammalian, plant, and bacterial cells that have been successfully transfected with a gene of interest. It acts by inhibiting protein synthesis and is commonly used in cell culture applications to identify and maintain cells that have been genetically modified.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (203 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (142 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (139 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (132 mentions)
Streptomycin, by Thermo Fisher Scientific (131 mentions)
Penicillin, by Thermo Fisher Scientific (130 mentions)
Puromycin, by Thermo Fisher Scientific (96 mentions)
L glutamine, by Thermo Fisher Scientific (92 mentions)
Hygromycin b, by Thermo Fisher Scientific (48 mentions)
Fetal bovine serum (fbs), by Merck Group (46 mentions)
Glutamax, by Thermo Fisher Scientific (46 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (41 mentions)
Puromycin, by Merck Group (38 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (38 mentions)
Zeocin, by Thermo Fisher Scientific (37 mentions)
Blasticidin, by Thermo Fisher Scientific (36 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (35 mentions)
Opti mem, by Thermo Fisher Scientific (33 mentions)
Hygromycin, by Thermo Fisher Scientific (31 mentions)
Hepes, by Thermo Fisher Scientific (28 mentions)
1 229 protocols using geneticin
1
Yellowtail Fin Cells Transfection
2
Stable cell line generation and characterization
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HeLa and U2OS cells were obtained from the Riken Cell Bank (RCB007, Tsukuba, Japan) and the Health Protection Agency (London, UK), respectively. HeLa cells were maintained in DMEM containing 10% calf serum (CS) and U2OS cells were in DMEM containing 10% fetal bovine serum (FBS). To generate HeLa cells stably expressing GFP-LacI (HeLa/GFP-LacI), they were transfected with plasmids encoding GFP-LacI using Effectene (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Cells were selected with 800 μg/ml Geneticin (11811-031, Life Technologies, Thermo Fisher Scientific, Tokyo, Japan). Several clones were isolated from the survivors and their expression levels were evaluated using Western blotting (WB) analysis. The selected clones were maintained in DMEM containing 10% FBS and 200 μg/ml Geneticin (11811-031, Life Technologies, Thermo Fisher Scientific, Tokyo, Japan). We established LEMD2 mutant cell lines (HeLa/mClover3-mAID-Lem2) expressing an mClover3-mAID-Lem2 protein as the sole Lem2 protein as described below (see section Depletion of NE proteins in Methods).
3
Doxycycline-inducible A9 Cell Lines
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Murine A9 and Human 293T cell lines were propagated as previously described [38] (link), [39] (link). Stable doxycycline-inducible A9 cell lines were generated by infection of A9 cells with pseudotyped virus using the pINDUCER20 lentiviral system [40] (link). Cell lines were selected with 800 µg/ml of geneticin (Gibco) and maintained like regular A9s except for addition of geneticin. A9 cells were parasynchronized in G0 by isoleucine deprivation as previously described [1] (link). pINDUCER20 lentiviral transformed cell lines were induced with 500 ng/mL doxycycline hydrochloride (MP Biomedical). MG132 (Calbiochem) was added at a final concentration of 10 µM.
4
U2OS Cell Line Culture Conditions
5
Stable Expression of Ackr3b and Ackr3
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COS cells, grown in Dulbecco modified Eagle's medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gibco Life Technologies), were transfected using Lipofectamin reagent (Invitrogen, Carlsbad, CA, USA) with a pcDNA vector harboring the ackr3b cDNA under the control of the CMV promoter and selected with geneticin (500 μg/ml; Invitrogen) to obtain ackr3b-COS cells. Stable expression of ackr3b was confirmed by RT-PCR. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and used at early (I–IV) passages. HUVECs were grown on culture plates coated with porcine gelatin in M199 medium (Gibco Life Technologies), supplemented with 20% FBS, endothelial cell growth factor (10 μg/ml), and porcine heparin (100 μg/ml) (Sigma-Aldrich). Chinese hamster ovary (CHO) cells were transfected with a bicistronic pIRES-EGFP vector harboring the human ACKR3 cDNA (kindly provided by Prof. Marcus Thelen, Institute for the Research Biomedicine, Bellinzona, Switzerland) using Lipofectamin reagent and selected with 350 μg/ml geneticin (Gibco Life Technologies) to obtain ACKR3-CHO cells. Stable expression of ACKR3 was confirmed by RT-PCR and fluorescent analysis of EGFP+ cells (see Figures 9A,B ).
6
U2OS Cell Line Culture Conditions
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All cell lines were cultured at 37°C in a 5% CO2 humidified atmosphere. U2OS cell lines stably expressing GFP47 (link), GFP-53BP1, RFP-53BP148 (link), GFP-UCHL5 (WT and DD), GFP-NFRKB and GFP-CtIP49 (link) were cultured with Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), 292 μg/ml L-Glutamine (Gibco) and 500 μg/ml Geneticin (Gibco). U2OS cells were cultured with identical media without Geneticin. The stable U2OS cell line expressing both GFP-EXO1 and monomeric version of Kusabira Orange2-fused human Geminin (1-110 a. a.) (mKO2-hGeminin) 50 (link) was cultured with the Geneticin containing media described above supplemented with 200 μg/ml Hygromycin B (Invitrogen). U2OS cells stably expressing the HR reporter Direct Repeat-GFP and U2OS cells carrying modified traffic light reporter based HR assay were cultured with DMEM containing FBS, penicillin, streptomycin, L-Glutamine and 1 μg/ml of puromycin (Sigma-Aldrich).
7
PODXL1 Knockout in PDAC Cell Lines
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PODXL1-knockout PDAC cell lines (for MIaPaCa-2, AsPC-1, and Panc-1) were generated using CRISPR/Cas9 system. Both plasmids, hCas9 (#41815 Addgene, Watertown, MA), and gRNA (guide RNA) Cloning Vector (#41824), were obtained from Addgene. The gRNA vector including PODXL1 target sequence (CGACACGATGCGCTGCGCGCtgg) located in part of the Exon 1 was prepared following the manufacturer’s instruction with “tgg” sequence as a Proto-spacer Adjacent Motif (PAM). The hCas9 and PODXL1 gRNA vector were cotransfected into cells using ViaFect™ Transfection Reagent (#E4981, Promega, Madison, WI). Twenty-four hours posttransfection, the cells were cultured with RPMI medium containing 500 μg/ml of Geneticin (#10131-35, Gibco, Thermo Fisher Scientific, Waltham, MA) for isolating the Geneticin-resistant clones. PODXL1-expression deficient clones from each PDAC line were confirmed by lack of PODXL1 protein, using immunoblot analysis with anti-PODXL1 antibody. Genetic mutation of PODXL1 in the knockout clone was also examined by genomic DNA sequencing of PCR-amplified product, using the specific primers for PODXL1-target sequence and the adjacent genomic DNA. The primers used for the sequencing were the following: Pod1.Ex1.check.Fw2: 5′-CAGCGGCAGGGAGGAAGAGC and Pod1.Ex1.check.Rv2 5′-GCGGTGCGGTCTCCCTTTTCTT.
8
Establishment of BHK-21 Cell Line Expressing Chikungunya Virus
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BHK-21 cells were transfected with the in vitro transcribed RNA. Briefly, 2 × 106 cells were resuspended in 100 μL of cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl2) with 2 mM ATP and 5 mM glutathione, and electroporation was performed in 2 mm cuvette with 140 V and 25 msec pulse (Gene Pulser Xcell, Bio-Rad, Hercules, CA, USA). Three days post-transfection cells were selected in medium supplemented with 700 μg/mL Geneticin® (Gibco, Waltham, MA, USA), and after ten days, cell colonies were removed by Scienceware® cloning discs (Sigma-Aldrich, St. Louis, MI, USA) soaked in trypsin (Gibco, Waltham, MA, USA), seeded individually and amplified in medium with Geneticin® (500 μg/mL). The selected cell line was denominated BHK-21-GLuc-nsP-CHIKV-99659.
9
Generating Stable EGFP-α-SynA53T Cell Line
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To create a stable cell line expressing EGFP-α-SynA53T, SH-SY5Y cells (ACC209, DSMZ; RRID: CVCL_0019) were transfected using Lipofectamine 2000 (Thermo Fisher). Cells were cultured in in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom) supplemented with 10% fetal bovine serum (FBS, GIBCO), 2 mM l -glutamine (GIBCO), and 1000 µg/ml geneticin for selection. Polyclonal cell lines were generated by fluorescence-activated cell sorting (FACS) with a BD FACS Aria III using FACSDiva 6.1.3 software. Upon selection, cells were cultured in medium supplemented with 200 µg/ml geneticin (Thermo Fisher) and penicillin/streptomycin (Thermo Fisher).
Cells were seeded as described25 (link) using 300 nM (monomer) of α-SynA53T PFFs or gold-conjugated WT α-Syn PFFs. In brief, sonicated PFFs were diluted in a mixture of 50 µl of Optimem (Biochrom) and 3 µl of Lipofectamine 2000. Subsequently, the suspension was added to 1 ml of cell culture medium.
Cells were seeded as described25 (link) using 300 nM (monomer) of α-SynA53T PFFs or gold-conjugated WT α-Syn PFFs. In brief, sonicated PFFs were diluted in a mixture of 50 µl of Optimem (Biochrom) and 3 µl of Lipofectamine 2000. Subsequently, the suspension was added to 1 ml of cell culture medium.
10
HEK-293 Cell Line Transfection Protocol
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The human embryonic kidney cell line HEK-293 (ATCC Certified from LGC Standards, Milan, Italy) was grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS).
5 × 106 HEK-293 cells, cultured overnight in 100 mm tissue culture dishes, were stably transfected with 10 μg of pPLR2.5, or pcDNA3 and 60 μl of LipofectAMINE (Invitrogen) for 5 h at 37°C (5% CO2). Transfected cells, named LR-293 and V-293 respectively, were selected by Geneticin (GIBCO) at 1.5 mg/ml, pooled, and cultured in the presence of 0.5 mg/ml Geneticin.
67LR highly expressing HT1080 fibrosarcoma [30 (link)] and MDAMB231 breast cancer cell lines [43 (link)] were grown in DMEM supplemented with 10% FBS.
5 × 106 HEK-293 cells, cultured overnight in 100 mm tissue culture dishes, were stably transfected with 10 μg of pPLR2.5, or pcDNA3 and 60 μl of LipofectAMINE (Invitrogen) for 5 h at 37°C (5% CO2). Transfected cells, named LR-293 and V-293 respectively, were selected by Geneticin (GIBCO) at 1.5 mg/ml, pooled, and cultured in the presence of 0.5 mg/ml Geneticin.
67LR highly expressing HT1080 fibrosarcoma [30 (link)] and MDAMB231 breast cancer cell lines [43 (link)] were grown in DMEM supplemented with 10% FBS.
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