U251‐MG cells were seeded in 24‐well plates at a density of 1 × 105 cells/well. 24 h later, the cells were transfected with PEI reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The total amount of 0.6 μg of plasmid DNA per well was used, including 0.4 μg of the luciferase‐coding reporter plasmid and 25 ng of Reg‐1, Reg‐2, TTP, corresponding RNase‐inactive mutant or the empty control plasmid. The quantity of DNA/well was normalized using an empty pcDNA3.1/mycHisA vector (Invitrogen). 24 h post‐transfection cells were stimulated with IL‐1β (Invivogen) or left untreated. 24 h after stimulation cells were lysed and firefly and Renilla luciferase activity were measured using Dual‐Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. For transfection efficiency normalization the Renilla luciferase, encoded on the same plasmid as the firefly luciferase (pmirGLO), was used. Luciferase data are presented as relative luciferase activity (firefly/Renilla) and normalized to 1.
Pei reagent
Manufactured by Thermo Fisher Scientific
PEI reagent is a polyethylenimine-based transfection agent used for the delivery of nucleic acids, such as DNA and RNA, into cells. It functions by forming complexes with the nucleic acids, which can then be taken up by the cells.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3.1 myc his a vector, by Thermo Fisher Scientific (2 mentions)
Il 1β, by InvivoGen (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions)
Pierce zeba desalting column, by Thermo Fisher Scientific (1 mentions)
Recombinant human n slit2, by Thermo Fisher Scientific (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
4 protocols using pei reagent
1
Luciferase Reporter Assay for U251-MG Cells
2
Large-scale Production of Bio-inactive N-SLIT2ΔD2
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Large-scale production of bio-inactive human N-SLIT2ΔD2 was performed by transfecting FreeStyle 293-F cells with human N-SLIT2ΔD2 cDNA (1 ug/mL) using PEI reagent (Polyethyleneimine, linear, M.W. 25,000, Thermo Fisher Scientific) (Patel et al., 2012 (link)). After 5 days, the culture medium was loaded onto HisPur Ni-NTA resin (Thermo Fisher Scientific), washed with imidazole (35 mM), eluted with imidazole (250 mM), and desalted using a Pierce Zeba desalting column (7 K MWCO; Thermo Fisher Scientific). Protein molecular weight was determined by immunoblotting using anti-6XHIS-HRP conjugated antibody. Protein activity was assayed by using a spreading assay in RAW264.7 cells, as described previously (Bhosle et al., 2020 (link)). Recombinant human N-SLIT2 was purchased from PeproTech (Cranbury, NJ, USA). Endotoxin levels in all preparations were measured using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA) and were less than 0.05 EU/ml.
3
Cell Culture and Transfection Protocols
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Human cervical cancer cell line HeLa and Nur77−/−HeLa53 (link) were cultured in RPMI-1640 (Sigma-Aldrich) with 10% fetal bovine serum (FBS), 100 U/ml penicillin-streptomycin (Sigma-Aldrich). HEK293T cells were maintained in DMEM (Sigma-Aldrich) containing 10% FBS and grown under standard tissue-culture conditions (37 °C, 5.0% CO2). Primary mouse embryonic fibroblasts (MEFs) and p62−/−MEFs were obtained from M.T.D.-M and J.M.’s lab73 (link). Transfection was performed using lipofectamine 2000 or PEI reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).
4
Investigating Reg Proteins in U251-MG Cells
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