Human MAO-A was stably over expressed in the human neuroblastoma SH-SY5Y cell line by cloning human MAO-A cDNA into the expression vector (pcDNA3.1-, Invitrogen, Karlsruhe, Germany), which was then transformed into competent bacteria. The expressed plasmid DNA was purified using DNA mini and DNA midi kits (Qiagen, Manchester, UK) and SH-SY5Y cells were transfected with 1 µg of pcDNA3.1(-) containing the hMAO-A insert or 1 µg of the empty pcDNA3.1(-) vector via electroporation using the Amaxa nucleofection system (Amaxa, Cologne, Germany). Electroporated SH-SY5Y cells were seeded in 6-well tissue culture plates in normal growth medium, which was replaced after 24 h. 48 h post-transfection, cells were passaged and seeded at 25% confluence in growth medium containing G418 sulphate (geneticin) at a concentration of 700 µg/ml. Stable SH-SY5Y clones were selected by their resistance to geneticin, which is conferred by the plasmid vector pcDNA3.1. The medium was replaced every 3–4 days until resistant foci were visible. Resistant cells were diluted in 96 well plates at a density of 0.5 cell/well and single clones were expanded and assayed for MAO-A expression.
Amaxa nucleofection system
Manufactured by Lonza
Sourced in Switzerland
The Amaxa nucleofection system is a laboratory equipment used for the transfection of cells. It utilizes an electrical pulse to facilitate the delivery of nucleic acids, such as DNA or RNA, into the cell nucleus. The core function of this system is to enable efficient gene transfer and expression in various cell types.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Human t cell nucleofection kit, by Lonza (2 mentions)
P3 primary cell 4d nucleofector x kit, by Lonza (2 mentions)
Pegfp n1, by Thermo Fisher Scientific (2 mentions)
Pcdna3, by Lonza (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Hypoxia chamber, by STEMCELL (1 mentions)
Profection kit, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Ha ubiquitin, by Addgene (1 mentions)
Pcdna mdm2, by Addgene (1 mentions)
Caakt, by Addgene (1 mentions)
Metafectene pro, by Biontex (1 mentions)
Microfuge, by Eppendorf (1 mentions)
Cell line nucleofector kit, by Lonza (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
Nucleofector kit 5, by Lonza (1 mentions)
U 015 program, by Lonza (1 mentions)
M199 medium, by Harvard Bioscience (1 mentions)
38 protocols using amaxa nucleofection system
1
Stable overexpression of human MAO-A in SH-SY5Y cells
2
Genetic Modification of Cell Lines
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The MWCL cell line was established and characterized at Mayo Clinic22 (link). The BCWM cell line was a kind gift from Dr. Steve Treon and the HBL-1 cell line was kindly provided by Dr. Thomas Witzig. Cell line authentication is described in Supplemental Methods . All cells lines were maintained in RPMI 1640 medium with 10–20% fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 µg/ml) were added. Cells were periodically checked for mycoplasma by PCR and were found to be negative. All cell lines were cultivated at 37 °C and 5% CO2. Transcription activator-like effector nuclease (TALENs) specific for targeting exon 5 of the TNFAIP3 gene were designed by the Mayo Clinic Genetics and Model Systems Core using the FusX system as previously described23 (link). Exon 5 was targeted because it is present in all reported TNFAIP3 isoforms. Cells were transfected with 10 μg of each TALEN vector arm by using the AMAXA® nucleofection system (Amaxa, Cologne, Germany). To track successful transfection, cells were co-transfected with a 2 μg GFP expressing plasmid. After 48 h, cells that have incorporated the GFP expression plasmids were isolated by single cell sorting at the Mayo Flow Cytometry Facility. Restriction enzyme digest was used to initially identify clones that carried the frameshift mutation.
3
KSHV and EBV Cell Line Cultivation
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PEL cell lines BCBL1 and HBL6 were maintained in RPMI1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml 12 streptomycin, 2 mM L-glutamine. Both cell lines are KSHV-positive; HBL6 are also EBV-positive. Cells were grown under either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) at 37°C for the indicated times and drug concentrations. Low O2 tension was obtained using either a Hypoxia Chamber (StemCell Technologies) or the INVIVO200 hypoxia workstation (Ruskinn Technology Ltd., UK). Transfection of BCBL1 cells was performed using the Amaxa nucleofection system (Amaxa, Cologne, Germany). Briefly, 5–10 × 106 cells were resuspended in nucleofector solution V prior to addition of DNA (CMV6-HA-myrAkt, CMV6 empty vector, siRNA to Akt 1 and 2 or scramble siRNA), then transferred to an Amaxa-certified cuvette and transfected using program T-001. Cells were examined for the expression of transfected genes 24 to 48 hours post transfection.
4
Regulation of Ucp1 Promoter Activity
5
Transfection and Protein Expression Optimization
6
Efficient Plasmid Transfection Protocols
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Cells were transfected with various plasmids or small interfering (si)RNAs using an Amaxa Nucleofection system (Amaxa Biosystems, Gaithersburg, MD) or Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturers' instructions. Detailed information on plasmid constructs are provided in the Supplementary Methods.
7
Silencing IL-6 in MDA-MB-231 Cells
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MDA-MB-231 cells were transfected with pSuperRetropuro-IL-6 shRNA 1 plasmid (Addgene) or endoribonuclease-prepared small interfering (esi)RNAs (Sigma EHU 048321) using an Amaxa Nucleofection system (Amaxa Biosystems, Gaithersburg, MD) or Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturers’ instructions. PSuperRetroPuro empty plasmid and esiFLUC (Firefly luciferase) were used as a control.
8
Transfection of HEK293T and Jurkat Cells
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HEK293T cells were maintained in DMEM medium and Jurkat cells in RPMI medium supplemented with 2 mM l -glutamine, 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% CO2 humidified atmosphere. HEK293T cells were transfected with indicated plasmids using METAFECTENE Pro (Biontex, Munich, Germany) according to the manufacturer's instructions. Jurkat cells were transfected with the constructs HIV-1JR-FLGag-iGFP and pCMV-CD63-mCherry or pCMV-CD63C145A,C146A-mCherry using the Amaxa nucleofection system (Amaxa Biosystems) as previously described56 (link).
9
Enhancing HUVEC Antioxidant Defenses
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HUVECs were transfected using the Amaxa nucleofection system (Amaxa, Gaithersburg, MD, USA) in procedures described by the manufacturer. Briefly, 1×106 cells were transfected with 2 μg of GPX1 or GPX4 expression vector DNA (Origene, MD, USA) or empty vector (as control, Origene, Rockville, MD, USA) per cuvette in 100 μL of HUVEC Nucleofector solution using the A034 setting on the nucleofector. Cells were supplemented with 100 nM sodium selenite and used between 1 and 3 days after Nucleofection.
10
Transfection and Infection of UT7/EpoS1 Cells
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UT7/EpoS1 cells were transfected by using the Amaxa Nucleofection System (Lonza, Basel, Switzerland), with V Nucleofector Reagent and T20 program setting, at a ratio of 1 µg insert DNA for 106 cells. Following transfection, the cells were incubated at 37 °C and 5% CO2 in complete medium at an initial density of 106 cells/mL, until collection at the indicated time points. Cells and cell-free supernatants were separated by centrifugation at 4000 rpm for 5 min in microfuge (Eppendorf, Hamburg, Germany), then fractions were used for analysis and/or successive infection experiments.
For infection experiments, cell-free supernatants obtained from transfected UT7/EpoS1 cells were added to EPCs cells at a ratio of 100 µL for 1 × 106 cells. Infection was carried out at 37 °C for 2 h, then cells were washed free of inoculum and expanded in complete medium at 37 °C and 5% CO2 at an initial density of 106 cells/mL, until collection at the indicated time points and subsequent processing as described.
For infection experiments, cell-free supernatants obtained from transfected UT7/EpoS1 cells were added to EPCs cells at a ratio of 100 µL for 1 × 106 cells. Infection was carried out at 37 °C for 2 h, then cells were washed free of inoculum and expanded in complete medium at 37 °C and 5% CO2 at an initial density of 106 cells/mL, until collection at the indicated time points and subsequent processing as described.
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