DENV-2 strain 16681 was propagated on C6/36 cells, as described previously [27 (link)]. Recombinant GFP-DENV was generated from the infectious clone pFK-DV-G2A strain 16681 (kind gift from Ralf Bartenschlager University of Heidelberg, [28 (link)]). The pFK-DV-G2A clone was propagated in E. coli strain D5α. Upon plasmid purification, the plasmid was linearized with XbaI (New England Biolabs, Ipswich, Massachusetts, USA) and capped RNA transcripts were synthesized by use of an SP6 polymerase (New England Biolabs). Viruses were harvested at 72 hours post-transfection (hpt) of RNA in BHK-21 cells via electroporation (Biorad Gene Pulser Xcell machine; 850 V, 25 μF, no resistance). Thereafter, GFP-DENV was propagated once by infecting C6/36 cells (MOI 0.1). Progeny virions were harvested, aliquoted and snap-frozen at 120 hours post-infection (hpi). WNV strain NY385-99 was a generous gift from Jaap Goudsmit (Crucell Holland BV) and was propagated on BHK-21 cells, as previously described [29 (link)]. All virus preparations were characterized, as described before [27 (link),30 (link)] by determination of the number of infectious particles by plaque assay on BHK-15 cells and the number of genome-equivalent particles by Q-RT-PCR. UV-inactivated GFP-DENV was obtained by exposure of the virus to UV-light for 4h. Inactivation was confirmed by plaque assay.
Gene pulser xcell machine
Manufactured by Bio-Rad
Sourced in Germany
The Gene Pulser Xcell machine is a laboratory equipment used for electroporation, a technique that introduces external DNA, RNA, or other molecules into cells by creating temporary pores in the cell membrane. The machine allows for precise control of the electrical parameters, such as voltage, capacitance, and pulse duration, to optimize the electroporation process for different cell types and applications.
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Lab products found in correlation
3 protocols using gene pulser xcell machine
1
Recombinant GFP-DENV Generation and Characterization
2
Electroporation of Dendritic and T-cells
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RNA electroporation (EP) was performed as described.29 (link) Centrifugation of DCs and T-cells was always performed for 10 min at 22°C and 149 g or 233 g, respectively. DCs were transfected with the RNA amounts indicated in the particular experiment. Prestimulated T-cells were electroporated26 (link) without RNA, 50 or 150 µg/ml trLT-RNA, 50 or 150 µg/ml trLT-DCL-RNA or 150 µg/ml of the control-DCL-RNA. For electroporation, cells were harvested in RPMI 1640, washed once in OptiMEM without phenol-red (Invitrogen, Karlsruhe, Germany), and then resuspended in OptiMEM with a maximal concentration of 6 × 107 DCs/ml or 12 × 107 T-cells/ml (all at room temperature). Electroporation was performed in 4 mm cuvettes (biolabproducts GmbH, Bebensee, Germany) with a Genepulser Xcell machine (Bio-Rad, Munich, Germany). The conditions were: square-wave pulse, 500 V, and 1 ms for DCs or 5 ms for T-cells, respectively.29 (link)After transfection, DCs were rested at 37°C for 4 h in DC medium supplemented with GM-CSF (800 IU/ml) and IL-4 (250 IU/ml), before using them for T-cell expansion or cryoconservation. Transfected T-cells were rested in T-cell medium for 1 h before being used for further experiments. The survival rate of the DCs was around 75% and over 50% when combined with cryoconservation. The survival rate of the T-cells was between 60–80%.
3
Transient Gene Overexpression by Lipofectamine and Electroporation
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Transient overexpression of the genes of interest was conducted using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transformation by electroporation was performed using a Gene Pulser Xcell machine (Bio-Rad) following the manufacturer's instructions.
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