Human ZIP4 cDNA encoding the long isoform of the protein and the Ala357 and Leu372 variants was cloned into pcDNA 3.1 (+) expression vector together with a hemagglutinin (HA) tag at the carboxyl terminus as described previously [71] (link). The Leu372Pro and Leu372Arg mutants, as well as the Thr357Ala and Leu372Val polymorphisms, were introduced via site-directed mutagenesis following standard conditions (QuikChange II XL; Stratagene; see Table S4 for complete human cDNA and primers used in the mutagenesis). The six human ZIP4 isoforms obtained (i.e. Ala357-Leu372, Ala357-Val372, Thr357-Leu372, Thr357-Val372 as well as Ala357-Pro372 and Ala357-Arg372) were confirmed by sequencing with the ABiPrism 3.1 BigDye kit before their use in transfection experiments. HeLa cells were cultured in DMEM plus 10% FBS and, subsequently, each of the various ZIP4 forms were transiently transfected using polyethyleneimine as the transfection reagent (PolySciences).
Transfection reagent
Manufactured by Polysciences
Sourced in United States
Polysciences' Transfection Reagent is a laboratory product used to facilitate the introduction of nucleic acids, such as DNA or RNA, into cells. The reagent enables efficient and reliable transfection, a process that is fundamental in various cell-based research applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyethyleneimine, by Polysciences (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Retrotek hiv 1 p24 antigen elisa kit, by ZeptoMetrix (1 mentions)
Prl null vector, by Promega (1 mentions)
Dual glo luciferase reporter system, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Cellstar tissue culture flasks, by Greiner (1 mentions)
Gene pulser cuvette, by Bio-Rad (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
6 protocols using transfection reagent
1
Cloning and Transfection of ZIP4 Isoforms
2
Retroviral and Lentiviral Vector Production
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Retroviral system plasmids pVSV-G, pCL-Eco and pMSCV-IRES-GFP were provided by Dr. Dario S. Zamboni (University of São Paulo, Brazil). To produce the pMSCV-Nef-IRES-GFP vector, the DNA fragment encoding NL4 Nef was transferred from pCIneo-Nef [30] (link) to pMSCV-IRES-GFP plasmid using the BglII and SalI restriction sites. Retroviruses expressing Nef and GFP or GFP alone were generated by cotransfection of PEAK cells (3×106) in a 100 mm tissue culture dish with: 3 µg pVSV-G, 6 µg pCL-Eco and 9 µg of pMSCV-IRES-GFP or pMSCV-Nef-IRES-GFP, using 55 µL of 25 kDa linear polyethylenimine (PEI) (1 µg/µl stock solution) transfection reagent (Polysciences Inc, Warrington, PA). Cell supernatants containing retroviruses were collected 36 h after transfection and stored at −80°C. HIV-1 luciferase reporter viruses were produced in HEK-293T cells. Briefly, 3×105 cells were transfected in 6 well/plate using pNL4.3 ΔNef Luc+ Env+ plasmid previously described [48] (link), using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. Supernatants containing viruses were collected 48 hours after transfection and stored at −80°C. HIV-1 titer was determined by p24 levels in supernatants using the RETROtek HIV-1 p24 antigen ELISA kit (ZeptoMetrix Corporation, Buffalo, NY).
3
BCL6 Transcriptional Regulation Assay
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HEK-293T cells were transiently transfected using polyethilenimine (PEI) transfection reagent (Polysciences Inc., Warrington, PA, USA) as previously described15 (link) and reporter experiments were performed essentially as described20 (link). 0.3 µg of the pGL3-basic vector (Promega) or 0.35 µg of the BCL6(exon1)-pGL3 reporter vector68 (link) and 0.1 µg of the pRL-null vector (Promega) were cotransfected with pCDNA-BCL6 expression vector15 (link). Cells were treated for 12 hours with different concentrations of romidepsin. Luciferase activities were measured 48 hours after transfection using the Dual-Glo Luciferase Reporter System (Promega, Madison, WI, USA) in a Luminometer TD 20/20 Turner Designs. For each determination, luciferase activity was calculated as the Firefly activity normalized by the Renilla activity. Luciferase activity in arbitrary units (a.u.) was shown as the increase in activation relative to the activity of the pGL3 vector alone and the maximum value for each condition was set to 100.
4
Polyethylenimine-Mediated Transient Transfection
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All transfections were carried out using linear polyethylenimine (PEI, MW 25,000) transfection reagent (#23966, Polysciences, Warrington, PA, USA). Cells were seeded in 24-well plates (4×104 cells/well) 24 h prior to transfection. Transfections were performed using 1 μg DNA at a PEI-DNA ratio of 3:1. F-MS2 and MCP-GFP plasmids were co-transfected at a ratio of 2:1.
5
Transient Transfection of Jurkat and COS-7 Cells
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Non-adherent cells: Jurkat T cells maintained at a logarithmic phase were washed in supplement-free RPMI 1640, resuspended either in 700 μl transfection buffer (1.8 g/ml sucrose, 2nM DTT in RPMI 1640) or in 250 μl Mirus Ingenio® electroporation solution and transferred into 0.4 cm-gap Gene Pulser cuvettes (BioRad) (20x106 cell/700 μl/cuvette). Plasmid DNA (10 μg/group, unless otherwise indicated) was added to the cuvettes, and electroporation was performed using a BioRad Gene Pulser (250V, 950mF). The cells were then cultured in 50 ml of complete RPMI 1640 in 145 mm tissue culture plates for 48 h. Unless otherwise indicated, all nucleofection experiments were carried out in triplicates using 3 separate dishes for each point.
Adherent cells: COS-7 cells maintained at a logarithmic phase were collected from culture dishes by trypsin treatment, washed in supplement-free DMEM, resuspended in 5 ml of 10% FCS-containing DMEM (2x106 cells/ml), and cultured in CellStar tissue culture flasks (Greiner Bio-one 690170). Within 24 hours of plating, adherent cells were transfected with the indicated DNA (5μg/group, unless otherwise indicated) using the PEI (Polysciences) transfection reagent in a 1:3 (DNA: PEI) ratio. The cells were then cultured in 10 ml complete DMEM in 75 cm2 tissue culture flasks for 48h.
Adherent cells: COS-7 cells maintained at a logarithmic phase were collected from culture dishes by trypsin treatment, washed in supplement-free DMEM, resuspended in 5 ml of 10% FCS-containing DMEM (2x106 cells/ml), and cultured in CellStar tissue culture flasks (Greiner Bio-one 690170). Within 24 hours of plating, adherent cells were transfected with the indicated DNA (5μg/group, unless otherwise indicated) using the PEI (Polysciences) transfection reagent in a 1:3 (DNA: PEI) ratio. The cells were then cultured in 10 ml complete DMEM in 75 cm2 tissue culture flasks for 48h.
6
Transient Transfection of HEK293T and LO2 Cells
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The human embryonic kidney cell line HEk293T and human normal liver cell line LO2 were cultured in DMEM (with glutamine) containing 10% FBS (BI) and 100 μg/mL Penicillin/Streptomycin. Polyethyleneimine (Polysciences) transfection reagent was used for transient transfection in all of these cells.
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