All HCC cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco BRL, NY). The 293FT cells used for viral packaging were cultured in high-glucose DMEM with 10% FBS, 6 mM L-glutamine, 1 mM sodium pyruvate (Invitrogen, USA), and 0.1 mM nonessential amino acids (NEAAs) (Invitrogen, USA). To maintain the expression of SV40 large T antigen in 293FT cells, geneticin at a dose of 500 μg/mL was also added (Roche, Germany). All the cells were kept at 37 °C in a humidified incubator containing 5% carbon dioxide.
Geneticin
Manufactured by Roche
Sourced in United States, Switzerland
Geneticin is a laboratory reagent used for selecting and maintaining mammalian cell lines that express recombinant genes. It is a potent antibiotic that inhibits protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
Pcdna3.1 expression vector, by Thermo Fisher Scientific (1 mentions)
Effectene, by Qiagen (1 mentions)
Xfect, by Takara Bio (1 mentions)
Pcgpv, by Cell Biolabs (1 mentions)
Prsv rev, by Cell Biolabs (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pcmv vsv g, by Cell Biolabs (1 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Lipofectamin 3000, by Thermo Fisher Scientific (1 mentions)
Facsaria flow cytometry, by BD (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
18 protocols using geneticin
1
Cell Culture Conditions for HCC and 293FT Cells
2
Sclerotinia sclerotiorum Cultivation and Pathogenicity
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The wild type isolate S. sclerotiorum “UF1” isolated from diseased petunia in Florida (Li et al., 2018 (link)) was used in this study. Strains were cultured, unless otherwise stated, on PDA medium at 25°C (Fan et al., 2017 (link)). S. sclerotiorum transformants were cultured on PDA amended with 100 μg/ml geneticin (Roche, Indianapolis, IN, United States) (Liu et al., 2018 (link)) and 50 μg/ml bromophenol blue (Sigma-Aldrich, United States) (Li et al., 2018 (link)). Liquid YPSU medium (50 ml containing yeast extract 4g, K2HPO4 1g, Mg2SO4 ⋅ 7H2O 0.5g, sucrose 15g, pH 6.5) was used to measure pH (Rollins, 2003 (link)). Common bean (Phaseolus vulgaris) used for pathogenicity assay grew in the greenhouse.
3
Evaluating CHD1L Tumorigenic Potential
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To evaluate the tumorigenic ability of CHD1L, the full-length CHD1L cDNA was amplified by PCR and cloned into a pcDNA3.1+ expression vector (Invitrogen) as described previously 8 (link). The expression plasmids were transfected into HO9810 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stable CHD1L expressing clones were selected using Geneticin (Roche), and the level of CHD1L expression was detected by Western blot analysis. Cells transfected with empty vector were used as controls.
4
Stable Expression of LBR Mutants
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5
Generating Stable Cell Lines with p27-2A-mRFP
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To produce lentivirus, pCSII-EF-mVenus-hGem, pCgpV (Cell Biolabs), pRsv-Rev (Cell Biolabs), and pCMV-VSVG (Cell Biolabs) were transfected to HEK293Ta cells. The supernatant was concentrated using an ultracentrifuge and added to mESCs. mVenus-hGemini expressing clone was selected using fluorescence. To establish the stable cell line of p27-2A-mRFP, pSMPUWneo-TRET-p27-TetOff was digested by XhoI, and the linearized plasmid was transfected into miPSCs [23 (link)] with Lipofectamine 2000 (Life Technologies). p27-2A-mRFP expressing clone was selected by resistance to Geneticin (100 μg/ml; Roche) at first, then selection was done using fluorescent active cell sorting (FACS). The stable cell line was designated as miPSCs-p27. Transient transfection of pSMPUW-TRET-p27-TetOff to miPSCs and mESCs was done with Xfect (Clontech), Lipofectamine 2000 (Life Technologies), or Lipofectamine 3000 (Life Technologies) transfection reagents.
6
Stable Expression of Mkl1 Variants in 4T1 Cells
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Full length Mkl1 (FL‐Mkl1) and the two Mkl1 mutants, mutB1‐Mkl1 comprising mutations in the B1 domain and ΔSAP‐Mkl1 lacking the SAP domain were previously described (Asparuhova et al., 2011 ). All Mkl1 variants were expressed as C‐terminal red fluorescent protein (RFP)‐tagged fusions. A control vector expressing RFP alone was described (Asparuhova et al., 2011 ).
4T1 mammary epithelial cells were grown in DMEM medium supplemented with 10% fetal calf serum (FCS, Invitrogen). In most of the experiments, cells were starved in 0.03% FCS/DMEM. To obtain 4T1 cells stably expressing FL‐Mkl1‐RFP (4T1‐FL), mutB1‐Mkl1‐RFP (4T1‐mutB1), ΔSAP‐Mkl1‐RFP (4T1‐ΔSAP) or RFP alone (4T1 control), cells were transfected using FuGENE® 6 (Roche) and selected with Geneticin (Roche) for 14 days before RFP‐based FACS sorting.
4T1 mammary epithelial cells were grown in DMEM medium supplemented with 10% fetal calf serum (FCS, Invitrogen). In most of the experiments, cells were starved in 0.03% FCS/DMEM. To obtain 4T1 cells stably expressing FL‐Mkl1‐RFP (4T1‐FL), mutB1‐Mkl1‐RFP (4T1‐mutB1), ΔSAP‐Mkl1‐RFP (4T1‐ΔSAP) or RFP alone (4T1 control), cells were transfected using FuGENE® 6 (Roche) and selected with Geneticin (Roche) for 14 days before RFP‐based FACS sorting.
7
Monoclonal Cell Line Generation
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All cells were cultured
in DMEM (Dulbecco’s
Modified Eagle Medium)/F12 (1:1) (Gibco) supplemented with 10% FBS
(fetal bovine serum, Gibco) and 1% penicillin/streptomycin at 37 °C
and 5% CO2. MDA-MB-231 cells were transfected using lipofectamin
3000 (ThermoFisher, L300001) following the manufacturer’s protocol
for a 6-well plate. After 24 h, the cell culture medium was supplemented
with 1800 μg/mL G418 (Geneticin, Roche), and cells were maintained
with G418 for all further experiments. After culturing the cells for
2 weeks with G418 selection, the transfected cells were sorted at
the core facility of the Institute of Molecular Biology (IMB) in Mainz
using BD FACS Aria III Cell sorter into a 96-well plate with one cell
per well. After expanding monoclonal cultures, their fluorescence
was measured again by flow cytometry (Attune NXT Acoustic Focusing
Cytometer, Invitrogen). The clones with the highest fluorescent signal
among all sorted cells were selected for future experiments.
in DMEM (Dulbecco’s
Modified Eagle Medium)/F12 (1:1) (Gibco) supplemented with 10% FBS
(fetal bovine serum, Gibco) and 1% penicillin/streptomycin at 37 °C
and 5% CO2. MDA-MB-231 cells were transfected using lipofectamin
3000 (ThermoFisher, L300001) following the manufacturer’s protocol
for a 6-well plate. After 24 h, the cell culture medium was supplemented
with 1800 μg/mL G418 (Geneticin, Roche), and cells were maintained
with G418 for all further experiments. After culturing the cells for
2 weeks with G418 selection, the transfected cells were sorted at
the core facility of the Institute of Molecular Biology (IMB) in Mainz
using BD FACS Aria III Cell sorter into a 96-well plate with one cell
per well. After expanding monoclonal cultures, their fluorescence
was measured again by flow cytometry (Attune NXT Acoustic Focusing
Cytometer, Invitrogen). The clones with the highest fluorescent signal
among all sorted cells were selected for future experiments.
8
Generating Stable KOr-Expressing K562 Cells
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K562 cells were obtained from the RIKEN Cell Bank (Tokyo, Japan) and pre-cultured in RPMI1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (Life Technologies), 100 U penicillin G/ml and 100 µg streptomycin (Sigma-Aldrich)/ml at 37°C in 5% CO2. The cells were transfected with the Kusabira-orange (KOr) fluorescent protein expression vector phKO1-MN1 (Amalgaam, Tokyo, Japan) using LipofectAMINE 2000 (Life Technologies) according to the manufacturer's instructions. Twenty-four hours after transfection, cells stably expressing KOr (K562-KOr cells) were selected in medium containing 800 µg geneticin/ml (Roche Diagnostics, Mannheim, Germany). After one week of culture, the KOr-expressing cells were purified by FACSAria flow cytometry (BD Biosciences, San Jose, CA) and further cultured.
9
Anti-CD20 Antibody Production in CHO Cells
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The transfection of pCOMIRES HIL anti-CD20 plasmid (encoding an anti-CD 20 (IgG) antibody, a secretable protein with molecular weight 150 kDa (two light chains, each with molecular weight 25 kDa, and two heavy chains, each with molecular weight 50 kDa)) into CHO cells was performed using a PolyPlus (JetPrime, New York, NY, USA) kit in six-well test plates (TPP, San Diego, CA, USA) according to the manufacturer’s instructions. The clones harboring the pCOMIRES HIL anti-CD20 transgene were selected from a mixed population by the single-cell dilution method. Geneticin (Roche, Gaillard, France) was used for selection at 800 μg/mL.
10
Overexpression of Adrenomedullin in Cells
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Full-length human ADM cDNA was amplified by PCR and cloned into the pEGFP-N1 expression vector (Clontech, Palo Alto, CA, USA) to construct pEGFP-N1-ADM, and then transfected into HuCCT1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with pEGFP-N1 were used as a negative control. Stable ADM-expressing clones were selected using geneticin (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 500 µg/ml.
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